Modified FGF-21 Polypeptides Comprising an Internal Deletion and Uses Thereof

ABSTRACT

Modified FGF-21 polypeptides and uses thereof are provided, for example, for the treatment of diseases associated with fibrosis. Modified FGF-21 polypeptides are disclosed that contain an internal deletion and optionally replacement peptide, optionally modified with at least one non-naturally-encoded amino acid, and/or optionally fused to a fusion partner.

RELATED APPLICATION DISCLOSURE

This application claims the benefit of U.S. Provisional Application Ser.No. 62/141,337 filed on Apr. 1, 2015; U.S. Provisional Application Ser.No. 62/141,383 filed on Apr. 1, 2015; U.S. Provisional Application Ser.No. 62/068,296 filed on Oct. 24, 2014; U.S. Provisional Application Ser.No. 62/068,523 filed on Oct. 24, 2014; U.S. Provisional Application Ser.No. 62/068,514 filed on Oct. 24, 2014; U.S. Provisional Application Ser.No. 62/068,534 filed on Oct. 24, 2014; and U.S. Provisional ApplicationSer. No. 62/068,526 filed on Oct. 24, 2014; each of which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING DISCLOSURE

This application includes as part of its disclosure a biologicalsequence listing in a file named “43270o2201.txt” and having a size of579,904 bytes, created on Oct. 22, 2015, which is hereby incorporated byreference in its entirety.

FIELD

This disclosure relates to modified FGF-21 polypeptides containing aninternal deletion that is optionally replaced by a peptide and the usesthereof for treatment or prevention of diseases and disorders.

BACKGROUND

Fibroblast growth factors are polypeptides widely expressed indeveloping and adult tissues (Baird et al., Cancer Cells, 3:239-243,1991) that play crucial roles in multiple physiological functions(McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 59:135-176, 1998;Burgess, W. H. et al., Annu. Rev. Biochem. 58:575-606 (1989). Accordingto the literature, the FGF family consists of at least twenty-twomembers (Reuss et al., Cell Tissue Res. 313:139-157 (2003)).

Fibroblast growth factor 21 (FGF-21) has been described in theliterature (Nishimura et al., Biochimica et Biophysica Acta,1492:203-206 (2000); WO 01/36640; and WO 01/18172, and U.S. PatentPublication No. 20040259780, each of which is incorporated by referenceherein in its entirety). Unlike other FGFs, FGF-21 has been reported notto have proliferative and tumorigenic effects (Ornitz and Itoh, GenomeBiology 2001, 2(3):reviews3005.1-3005.12).

Certain FGF-21 polypeptides and uses thereof are described in U.S.Patent Publication No. 20010012628, U.S. Pat. No. 6,716,626, U.S. PatentPublication No. 2004/0259780, WO 03/011213, Kharitonenkov et al. J ClinInvest. 2005 June; 115(6):1627-35, WO 03/059270, U.S. Patent PublicationNo. 2005/0176631, WO 2005/091944, WO 2007/0293430, U.S. PatentPublication No. 2007/0293430, WO/2008/121563, U.S. Pat. No. 4,904,584,WO 99/67291, WO 99/03887, WO 00/26354, and U.S. Pat. No. 5,218,092 eachof which is incorporated by reference herein in its entirety.

Human FGF-21 has been reported to have a propensity to undergoproteolysis in vivo, form aggregates in vitro, undergo deamidation(Gimeno and Moller, Trends Endocrinol Metab. 2014 June; 25(6):303-11;U.S. Pat. No. 8,361,963; Hecht et al., PLoS One. 2012; 7(11):e49345;U.S. Patent Publication No. 2007/0293430; WO 2006/0065582), potentiallylimiting the shelf-life of pharmaceutical compositions containingFGF-21. Aggregates and deamidated forms of therapeutic polypeptides maypotentially increase immunogenicity (see U.S. Department of Health andHuman Services, “Immunogenicity Assessment for Therapeutic ProteinProducts,” August 2014; Subramanyam (ed.), “Therapeutic ProteinImmunogenicity Focus Group Newsletter,” American Association ofPharmaceutical Scientists, Vol. 1, Issue 3 (December 2011)).

Prior work published as WO 2008/121563 and U.S. Patent Publication No.2008/0255045 demonstrated that certain human FGF-21 polypeptidesmodified to contain a non-naturally encoded amino acid linked topoly(ethylene glycol) at specified positions exhibited increased in vivohalf-life and/or retained biological activity. The exemplified humanFGF-21 polypeptides did not, however, include sequence deletions orsubstitutions described herein.

Fibrosis is the formation of excess fibrous connective tissue in anorgan or tissue. Excess deposition of fibrous tissue is associated withpathological conditions that can lead to impairment of organ or tissuefunction. Affected organs can include the lungs (lung or pulmonaryfibrosis), liver (liver or hepatic fibrosis), kidney (kidney or renalfibrosis), and heart (cardiac fibrosis). Fibrosis can also affect othertissues and organs including joints, skin, intestine, bone marrow, andothers. Exemplary fibrotic conditions or diseases include, but are notlimited to, nonalcoholic steatohepatitis (NASH), which affects theliver; diabetic kidney disease and diabetic nephropathy, which affectthe kidney; and metabolic heart failure, which affects the heart. Forexample, NASH is characterized by fat, inflammation and damage in theliver in people who consume little or no alcohol and can lead to livercirrhosis. NASH tends to be diagnosed in overweight or obese middle-agedpeople who often have elevated blood lipid levels and diabetes orprediabetes.

Embodiments of the present invention address, among other things,problems associated with the activity and production of FGF-21polypeptides, the production of an FGF-21 polypeptide with improvedbiological or pharmacological properties, such as improved therapeutichalf-life, and methods of treating or preventing diseases and disorders.

SUMMARY

Provided herein are modified FGF-21 polypeptides comprising apolypeptide having an amino acid sequence selected from SEQ ID NOs: 1-7,except that said amino acid sequence comprises: (i) an internal deletionof between 2 and 19 amino acids (such as between 5 and 19 amino acids),wherein said internal deletion is within a region corresponding to aminoacids 116 to 134 of SEQ ID NO:1, wherein said internal deletion isreplaced by a replacement peptide having a length of between 0-12 aminoacids; and (ii) 9 or fewer additional amino acid substitutions,deletions, and/or insertions.

Also provided herein are compositions comprising any of the modifiedFGF-21 polypeptides described herein and a pharmaceutically acceptablecarrier or excipient.

Provided herein are modified FGF-21 polypeptides comprising apolypeptide having an amino acid sequence selected from SEQ ID NOs: 1-7,except that said amino acid sequence comprises: (i) an internal deletionof between 2 and 19 amino acids (such as between 5 and 19 amino acids),wherein said internal deletion is within a region corresponding to aminoacids 116 to 134 of SEQ ID NO:1, wherein said internal deletion isreplaced by a replacement peptide having a length of between 0-12 aminoacids; and (ii) 9 or fewer additional amino acid substitutions,deletions, and/or insertions; and (iii) a fusion partner.

Also provided herein are methods of regulating at least one of glucoseand lipid homeostasis, glucose uptake, GLUT 1 expression, and/or serumconcentrations of glucose, triglycerides, insulin or glucagon in apatient in need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedescribed herein.

Also provided herein are methods of increasing insulin sensitivity,increasing the level of adiponectin, reducing the level of bloodglucose, reducing the level of glucagon, reducing the level oftriglyceride, reducing the level of fructosamine, reducing the level oflow density cholesterol, or reducing the level of C-reactive protein ina patient in need thereof or in a sample of blood, serum, or anothersample of said patient, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedescribed herein.

Also provided herein are methods of treating a condition or disorderselected from obesity, diabetes, pancreatitis, insulin resistance,hyperinsulinemia, glucose intolerance, hyperglycemia, metabolicsyndrome, impaired glucose tolerance, inadequate glucose clearance, highblood glucose, Type A Insulin Resistance, Type C Insulin Resistance (AKAHAIR-AN Syndrome), Rabson-Mendenhall Syndrome, Donohue's Syndrome orLeprechaunism, hyperandrogenism, hirsuitism, or acanthosis nigricans,and Prader-Willi syndrome in a patient in need thereof, comprisingadministering to the patient a therapeutically effective amount of amodified FGF-21 polypeptide described herein.

Also provided herein are methods of treating type 1 diabetes, type 2diabetes or obesity in a patient in need thereof, comprisingadministering to the patient a therapeutically effective amount of amodified FGF-21 polypeptide described herein.

Also provided herein are methods of treating a disease associated withfibrosis comprising administering to a patient in need thereof aneffective amount of a modified FGF-21 polypeptide described herein.

Also provided herein are methods of treating a disease associated withfibrosis comprising administering to a patient in need thereof aneffective amount of a composition comprising the modified FGF-21polypeptide described herein and a pharmaceutically acceptable carrieror excipient.

Also provided herein are methods of treating liver fibrosis or cirrhosisin a patient in need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedescribed herein.

Also provided herein are methods of treating or preventing NASH in apatient in need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedescribed herein.

Also provided herein are methods of treating NASH and/or liver fibrosis,comprising administering to a patient in need thereof an effectiveamount of a composition comprising the modified FGF-21 polypeptidedescribed herein and a pharmaceutically acceptable carrier or excipient.

Also provided herein are methods of decreasing the hepatic fat fractionin a patient in need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedescribed herein, wherein optionally said patient is at risk ofdeveloping or has been diagnosed with NASH.

Also provided herein are methods of increasing adiponectin levels (suchas plasma total adiponectin and/or high molecular weight adiponectin) ina patient in need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedescribed herein, wherein optionally said patient is at risk ofdeveloping or has been diagnosed with NASH.

Also provided herein are methods of treating heart failure or cardiacfibrosis in a patient in need thereof comprising administering to thepatient a therapeutically effective amount of a modified FGF-21polypeptide described herein.

Also provided herein are methods of treating kidney or renal fibrosis ina patient in need thereof comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedescribed herein.

Also provided herein are methods of treating lung fibrosis in a patientin need thereof comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedescribed herein.

Also provided herein are methods of treating a disease associated withfibrosis in a patient in need thereof, comprising administering to thepatient an effective amount of a modified FGF-21 polypeptide comprisingone or more non-naturally encoded amino acids, wherein said modifiedFGF-21 polypeptide possesses at least 90% identity to a human FGF-21polypeptide having an amino acid sequence selected from SEQ ID NOs:1-7and 201, wherein said disease associated with fibrosis is selected fromNASH, liver fibrosis, diabetic kidney disease, chronic kidney disease,renal fibrosis, lung fibrosis, cardiac fibrosis, heart failure, andmetabolic heart failure.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A-B. Exemplary modified FGF-21 polypeptide sequences. Coordinatesindicated are relative to the wild-type FGF-21 polypeptide of SEQ IDNO:1. The full polypeptides each include the N-terminal sequenceMHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY, followed by the indicatedamino acid at position 108, followed by the indicated sequence of theregion labeled “Amino Acids 109-149”, followed by the sequencePGILAPQPPDVGSSDPLSM, followed by the indicated sequence of the regionlabeled “Amino Acids 169-181.” The full amino acid sequences of themodified FGF-21 polypeptides are also shown in Example 4, below.

FIG. 2. Exemplary dose response curve for in vitro FGF-21 activity(measured by FGF21-dependent phosphorylation of extracellularsignal-regulated kinase (ERK) 1/2 in a cell-based assay) for PegylatedCompound 1 and Pegylated Compound 2.

FIG. 3. Representative results of differential scanning calorimetry(DSC) performed for evaluation of thermal stability of modified FGF-21polypeptides. In this example, Pegylated Compound 2 was shown to have atransition midpoint (“Tm”) temperature approximately 8 degrees C. higherthan Pegylated Compound 1. Thermal reversibility was >95% (not shown).

FIG. 4. Representative results of thermal scanning fluorescence (TSF)performed for evaluation of thermal stability of modified FGF-21polypeptides. In this figure, Compound 10 was shown to have a transitionmidpoint (“Tm”) temperature approximately 8 degrees C. higher thanCompound 1 (having a wild-type FGF-21 sequence except that an N-terminalmethionine included for expression in E. coli).

FIG. 5. Left Panel: Measurement of deamidation over time (indicated bycharge heterogeneity) at 25 degrees C. for Pegylated Compound 1 andPegylated Compound 2. Pegylated Compound 2 exhibited decreased chargeheterogeneity formation, indicative of greatly slowed deamidation. RightPanel: Measurement of aggregate formation over time (measured by sizeexclusion chromatography) at 40 degrees C. for Pegylated Compound 1 andPegylated Compound 2 each in Sucrose/Histidine buffer at pH 7.0 and aprotein concentration of 7.5 mg/mL. Pegylated Compound 2 exhibiteddecreased aggregate formation. Together, these results are predictive ofgreater shelf stability and ability to be formulated to a higherconcentration for Pegylated Compound 2 relative to Pegylated Compound 1.

FIG. 6. Measurement of high molecular weight (HMW) aggregate formationover time for modified FGF-21 polypeptides at pH 6.5 insucrose/histidine buffer (upper panel), pH 7.0 in sucrose/histidinebuffer (middle panel), and pH 8.3 in sucrose/TRIS buffer (lower panel).HMW aggregate formation was more pronounced at pH 6.5 and pH 7.0 than atpH 8.3. The majority of FGF-21 variants exhibited decreased HMWaggregate formation relative to Pegylated Compound 1, though for a fewcompounds HMW aggregate formation was similar to or higher than forCompound 1,

FIG. 7. Measurement of deamidation propensity (indicated by formation ofacidic variants over time) for modified FGF-21 polypeptides at pH 6.5 insucrose/histidine buffer (upper panel), pH 7.0 in sucrose/histidinebuffer (middle panel), and pH 8.3 in sucrose/TRIS buffer (lower panel).Compared to Pegylated Compound 1, all compounds shown exhibiteddecreased charge acidic variant formation, indicating decreaseddeamidation.

FIG. 8. Measurement of solubility for modified FGF-21 polypeptides.Relatively lower formation of high molecular weight aggregates at agiven polypeptide concentration is indicative of greater solubility,which would result in the ability to be formulated to a greaterconcentration. Observations of protein concentration vs. percentfraction of High Molecular Weight (HMW) species of tested compoundsdetermined via the plug flow filtration assay in PBS buffer at pH 7.2.The linearized slope of the line fit to each of these observations wasused as an estimate of protein aggregation propensity.

FIG. 9. Percentage of donors with a CD4+ T cell proliferation responsefollowing a 7 day protein and peripheral blood mononuclear cell (PBMC)incubation. The control proteins are E. coli expressed proteins withknown T cell activation properties. Comparison of non-pegylated modifiedFGF-21 compounds to Compound 1 (having a wild-type FGF-21 sequenceexcept that an N-terminal methionine included for expression in E.coli). “ConA” is an abbreviation for Concanavalin A, a known immunogenicprotein.

FIG. 10. Pharmacokinetic analysis of Pegylated Compound 1 and PegylatedCompound 2 after a single S.C. administration of 0.05 mg/kg to ob/obmice. Levels of the modified FGF-21 compounds were measured as both thetotal and C-terminally intact (active) polypeptides. Pegylated Compound2 exhibited a much greater total AUC for the C-terminally intact formthan Pegylated Compound 1, indicating greatly reduced in vivoproteolysis of Pegylated Compound 2. Results are shown graphically inthe upper panel and pharmacokinetic parameters are tabulated in thelower left panel (Pegylated Compound 1) and lower right panel (PegylatedCompound 2).

FIG. 11A-B. Pharmacokinetic analysis of Pegylated Compound 1 andPegylated Compound 2 after administration to cynomolgus monkeys. Levelsof the modified FGF-21 compounds were measured as both the (A) total and(B) C-terminally intact (active) polypeptides. Pegylated Compound 2exhibited a much greater total AUC for the C-terminally intact form thanPegylated Compound 1, indicating greatly reduced in vivo proteolysis ofPegylated Compound 2.

FIG. 12. Change in glycated hemoglobin (HbA1c) vs. vehicle on Day 21 ofa repeated dosing study in ob/ob mice.

FIG. 13. Body weight over 21-days of a repeated dosing study in ob/obmice.

FIG. 14. Percent change in body weight over 21-days of a repeated dosingstudy in ob/ob mice.

FIG. 15. Plasma insulin concentrations during a 21-day repeated dosingstudy in ob/ob mice.

FIG. 16. Plasma glucose concentrations during a 21-day repeated dosingstudy in ob/ob mice.

FIG. 17. Change in AUC plasma glucose concentrations during a 21-dayrepeated dosing study in ob/ob mice, expressed as the percentagedifference from vehicle-treated controls.

FIG. 18. Plasma triglyceride concentrations during a 21-day repeateddosing study in ob/ob mice.

FIG. 19. Body weight changes in a Stelic NASH mouse study.

FIG. 20. Total food consumption in a Stelic NASH mouse study.

FIG. 21. Body weight in a Stelic NASH mouse study.

FIG. 22. Liver weight in a Stelic NASH mouse study.

FIG. 23. Liver-to-body weight ratio in a Stelic NASH mouse study.

FIG. 24. Whole blood glucose in a Stelic NASH mouse study.

FIG. 25. Plasma ALT in a Stelic NASH mouse study.

FIG. 26. Plasma triglyceride in a Stelic NASH mouse study.

FIG. 27. Plasma total cholesterol in a Stelic NASH mouse study.

FIG. 28. Liver triglyceride in a Stelic NASH mouse study.

FIG. 29. Liver cholesterol in a Stelic NASH mouse study.

FIG. 30A-B. Representative photomicrographs of HE-stained liver sectionsin a Stelic NASH mouse study.

FIG. 31. NAFLD Activity score in a Stelic NASH mouse study.

FIG. 32A. Steatosis score in a Stelic NASH mouse study.

FIG. 32B. Lobular inflammation score in a Stelic NASH mouse study.

FIG. 32C. Hepatocyte ballooning score in a Stelic NASH mouse study.

FIG. 33A-B. Representative photomicrographs of Sirius red-stained liversections in a Stelic NASH mouse study.

FIG. 34. Fibrosis area in a Stelic NASH mouse study.

FIG. 35A-B. Representative photomicrographs of F4/80-immunostained liversections in a Stelic NASH mouse study.

FIG. 36 Inflammation area in a Stelic NASH mouse study.

FIG. 37A-B. Representative photomicrographs of Oil red-stained liversections in a Stelic NASH mouse study.

FIG. 38. Fat deposition area in a Stelic NASH mouse study.

FIG. 39A-D. Relative gene expression in a Stelic NASH mouse study.Expression results for (A) Alpha-SMA, (B), TIMP-1, (C) Collagen Type 1,and (D) TGF-β.

FIG. 40A-D. Exemplary modified FGF-21 polypeptide sequences fused to anFc domain, a PIKE adnectin or human serum albumin polypeptide. “FusionArrangement” describes the general orientation of the fusion protein,from the N- to C-terminus, which is intended to illustrate, but not tolimit, the corresponding polypeptide sequence. HuSA(C34A): a modifiedhuman serum albumin having the amino acid sequence of SEQ ID NO:321);HuSA(C34A; des Leu-585): a modified human serum albumin having the aminoacid sequence of SEQ ID NO:322; PKE(1): a PKE adnectin having the aminoacid sequence of SEQ ID NO:319; PKE(2): a PKE adnectin having the aminoacid sequence of SEQ ID NO:320. L followed by a number indicates alinker; FGF-21(Cmp. 2) indicates an FGF-21 sequence containing aninternal deletion and replacement peptide (specifically, Compound 2) andFGF-21(Cmp. 1) indicates an FGF-21 sequence lacking said deletion andreplacement peptide (specifically, Compound 1). FGF-21(Cmp. 1) andFGF-21(Cmp. 2) may include or lack the N-terminal methionine containedin Compounds 1 and 2. Variant forms of Fc polypeptide sequences areidentified parenthetically, e.g., Fc(hIgG1a_191). The notationsFGF-21-1aa(Cmp. 2) and FGF-21-3aa(Cmp. 2) refer to the Compound 2sequence modified by deletion of the recited number of amino acids (1 or3, respectively) from its C-terminus.

FIG. 41. Measurement of solubility for modified FGF-21 polypeptidesfused to an adnectin fusion partner. Relatively lower formation of highmolecular weight aggregates at a given polypeptide concentration isindicative of greater solubility, which would result in the ability tobe formulated to a greater concentration. Observations of proteinconcentration vs. percent fraction of High Molecular Weight (HMW)species of tested compounds determined via the plug flow filtrationassay in PBS buffer at pH 7.2. The linearized slope of the line fit toeach of these observations was used as an estimate of proteinaggregation propensity.

FIG. 42A-B. Body weight of treatment groups in a Stelic NASH mousestudy. A. Comparison of mice treated with vehicle or PEG-Compound(“PEG-Cmpd”) 1 for weeks 9-12. B. Comparison of mice treated withvehicle or PEG-Compound 1 for weeks 9-15.

FIG. 43A-B. Liver weight of treatment groups in a Stelic NASH mousestudy. A. Comparison of mice treated with vehicle or PEG-Compound 1 forweeks 9-12. B. Comparison of mice treated with vehicle or PEG-Compound 1for weeks 9-15. Liver weight was significantly decreased (p<0.001) formice treated over weeks 9-15.

FIG. 44A-B. Liver-to-body weight ratio of treatment groups in a StelicNASH mouse study. A. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-12. B. Comparison of mice treated withvehicle or PEG-Compound 1 for weeks 9-15. Liver-to-body weight ratio wassignificantly decreased (p<0.01 and p<0.001, respectively) for micetreated over weeks 9-12 or weeks 9-15.

FIG. 45A-B. Right kidney weight of treatment groups in a Stelic NASHmouse study. A. Comparison of mice treated with vehicle or PEG-Compound1 for weeks 9-12. B. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-15.

FIG. 46A-B. Left kidney weight of treatment groups in a Stelic NASHmouse study. A. Comparison of mice treated with vehicle or PEG-Compound1 for weeks 9-12. B. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-15.

FIG. 47 A-B. Whole blood glucose of treatment groups in a Stelic NASHmouse study. A. Comparison of mice treated with vehicle or PEG-Compound1 for weeks 9-12. B. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-15. Whole blood glucose was significantlydecreased (p<0.05) for mice treated over weeks 9-15.

FIG. 48A-B. Plasma ALT of treatment groups in a Stelic NASH mouse study.A. Comparison of mice treated with vehicle or PEG-Compound 1 for weeks9-12. B. Comparison of mice treated with vehicle or PEG-Compound 1 forweeks 9-15. Plasma ALT was significantly decreased (p<0.01) for micetreated over weeks 9-15.

FIG. 49A-B. Plasma triglyceride of treatment groups in a Stelic NASHmouse study. A. Comparison of mice treated with vehicle or PEG-Compound1 for weeks 9-12. B. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-15.

FIG. 50A-B. Plasma total cholesterol of treatment groups in a StelicNASH mouse study. A. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-12. B. Comparison of mice treated withvehicle or PEG-Compound 1 for weeks 9-15.

FIG. 51A-B. Liver triglyceride of treatment groups in a Stelic NASHmouse study. A. Comparison of mice treated with vehicle or PEG-Compound1 for weeks 9-12. B. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-15. Liver triglycerides were significantlydecreased (p<0.01 and p<0.001, respectively) for mice treated over weeks9-12 or weeks 9-15.

FIG. 52A-B. Liver cholesterol of treatment groups in a Stelic NASH mousestudy. A. Comparison of mice treated with vehicle or PEG-Compound 1 forweeks 9-12. B. Comparison of mice treated with vehicle or PEG-Compound 1for weeks 9-15. Liver cholesterol was significantly decreased (p<0.01and p<0.001, respectively) for mice treated over weeks 9-12 or weeks9-15.

FIG. 53A-H. Representative photomicrographs of HE-stained liver sectionsof mice treated for weeks 9-12 with vehicle (A-B) or PEG-Compound 1(C-D), and for weeks 9-15 with vehicle (E-F) or PEG-Compound 1 (G-H).

FIG. 54A-B. NAFLD activity score of treatment groups in a Stelic NASHmouse study. A. Comparison of mice treated with vehicle or PEG-Compound1 for weeks 9-12. B. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-15. NAFLD activity score was significantlydecreased (p<0.01 and p<0.001, respectively) for mice treated over weeks9-12 or weeks 9-15.

FIG. 55A-H. Representative photomicrographs of Sirius red-stained liversections of mice treated for weeks 9-12 with vehicle (A-B) orPEG-Compound 1 (C-D), and for weeks 9-15 with vehicle (E-F) orPEG-Compound 1 (G-H).

FIG. 56A-B. Summary of liver fibrosis area of treatment groups in aStelic NASH mouse study. A. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-12. B. Comparison of mice treated withvehicle or PEG-Compound 1 for weeks 9-15. Fibrosis area wassignificantly decreased (p<0.05) for mice treated with PEG-Compound 1for weeks 9-15.

FIG. 57A-H. Representative photomicrographs of F4/80-immunostained liverof mice treated for weeks 9-12 with vehicle (A-B) or PEG-Compound 1(C-D), and for weeks 9-15 with vehicle (E-F) or PEG-Compound 1 (G-H).

FIG. 58A-B. Summary of inflammation area of treatment groups in a StelicNASH mouse study. A. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-12. B. Comparison of mice treated withvehicle or PEG-Compound 1 for weeks 9-15.

FIG. 59A-H. Representative photomicrographs of Oil red-stained liversections of mice treated for weeks 9-12 with vehicle (A-B) orPEG-Compound 1 (C-D), and for weeks 9-15 with vehicle (E-F) orPEG-Compound 1 (G-H).

FIG. 60A-B. Summary of fat deposition area of treatment groups in aStelic NASH mouse study. A. Comparison of mice treated with vehicle orPEG-Compound 1 for weeks 9-12. B. Comparison of mice treated withvehicle or PEG-Compound 1 for weeks 9-15. Fat deposition area wassignificantly decreased for mice treated over weeks 9-12 or weeks 9-15(both p<0.001).

FIG. 61. Serum total adiponectin measurements in a Stelic NASH mousestudy. Serum total adiponectin was significantly increased in micetreated with PEG-Compound 1 at 3.0 mg/kg, compared to vehicle-treatedmice, in terminal plasma samples from mice treated between weeks 5-9 orweeks 7-9 (p<0.001, Dunnett's TTEST).

FIG. 62. Serum total adiponectin measurements in a Stelic NASH mousestudy. Serum total adiponectin was significantly increased in micetreated with PEG-Compound 1 at 3.0 mg/kg, compared to vehicle-treatedmice, in terminal plasma samples from mice at week 12 or 15 (p<0.05,Dunnett's TTEST).

FIG. 63. Concentration-response curves for three FGF21 variants:N-terminally His-tagged Compound 1 (His-Cmpd 1), PEG-Compound 1(PEG-Cmpd 1) and PEG-Compound 2 (PEG-Cmpd 2) in the 5 h Elk1-luciferaseassay. Values are from 6 independent experiments with 3 or 4 replicatesper experiment. Z′ values varied between 0.6-0.9 indicating acceptableassay quality (Z′>0.5).

FIG. 64. Potency of FGF-21 variants measured in the cell-basedElk1-luciferase assay. Each data point indicates an individual replicateand the horizontal bar represents the mean value. Values are expressedas the base 10 logarithm of the measured EC₅₀ value in moles.

FIG. 65. Effect of single, acute doses of PEG-Compound 2 on percentchange in body weight in C57BL/6J mice. C57BL/6J mice were treated witheither vehicle (250 mM Sucrose/20 mM Tris, pH 8.3), or PEG-Compound 2 at0.03, 0.1, 0.3, or 1.0 mg/kg, n=10/group. Percent change in body weightsin C57BL/6J mice at baseline (0), 24, 48, 72 and 144 h post SC dose wasdetermined. All values are mean±SEM *P<0.05, ***P<0.001, ****P<0.0001vs. vehicle, (one-way ANOVA followed by Dunnett's test).

FIG. 66A. Effect of single, acute doses of PEG-Compound 2 on plasmatotal adiponectin in C57BL/6J mice. Plasma total adiponectinconcentration at baseline (0), 24, 48, 72, and 144 h post SC dose wasdetermined. Non-fasted C57BL/6J mice were treated with either vehicle(250 mM Sucrose/20 mM Tris, pH 8.3), or PEG-Compound 2 at 0.03, 0.1,0.3, or 1.0 mg/kg, n=10/group. All values are mean±SEM **P<0.01,***P<0.001, **** P<0.0001 vs. vehicle, (one-way ANOVA followed byDunnett's).

FIG. 66B. Effect of single, acute doses of PEG-Compound 2 on percentchange in plasma total adiponectin in C57BL/6J mice. C57BL/6J mice weretreated with either vehicle (250 mM Sucrose/20 mM Tris, pH 8.3), orPEG-Compound 2 at 0.03, 0.1, 0.3, or 1.0 mg/kg, n=10/group. Plasma totaladiponectin concentration was determined at baseline 24, 48, 72, and 144h post SC dose, and percent change relative to baseline was determined.All values are mean±SEM **P<0.01, ***P<0.001, **** P<0.0001 vs. vehicle,(one-way ANOVA followed by Dunnett's test).

FIG. 67A. Effects of FGF-21 variants on plasma total adiponectin inC57BL/6J mice. Both PEG-Compound 2 and PEG-Compound 20 treatmentresulted in significant increases in total plasma adiponectin 6 dayspost-dose at both 0.3 and 1 mpk doses (*P<0.05, **P<0.01, ***P<0.001).

FIG. 67B. Effects of FGF-21 variants on plasma total adiponectin inC57BL/6J mice. The 54.3 nmol/kg dose of PEG-Compound 2, Compound 105,and Compound 112 significantly increased the percentage change in totaladiponectin as compared to the percentage change in total adiponectin invehicle-treated controls on Days 3 and 6.

FIG. 67C. Effects of FGF-21 variants on plasma total adiponectin inC57BL/6J mice. Both 18.1 and 54.3 nmol/kg doses of Compound 182significantly increased the percentage change in plasma totaladiponectin (as compared to the percentage change observed in thevehicle controls) on Days 3 and 6.

FIG. 67D. Effects of FGF-21 variants on plasma total adiponectin inC57BL/6J mice. Both Compound 171 and Compound 181 significantlyincreased the percentage change from baseline plasma total adiponectinon Days 3 and 6 post-dose, in a dose-dependent manner, compared to thevehicle-treated control.

FIG. 67E. Effects of FGF-21 variants on plasma total adiponectin inC57BL/6J mice. The percentage change from baseline in plasma totaladiponectin (as compared to vehicle) was significantly increased byCompound 170 on Days 3 and 6 at 18.1 nmol/kg and 54.3 nmol/kg.

FIG. 67F. Effects of FGF-21 variants on plasma total adiponectin inC57BL/6J mice. The 10 mg/kg dose of PEG-Compound 1 significantlyincreased the percentage change from baseline in plasma totaladiponectin as compared to vehicle-treated controls on Days 3 and 6.

FIG. 68. Effect of PEG-Compound 2 on Albuminuria (Urine ACR). # P<0.05,disease (unix db/db, Vehicle, n=9-10) versus normal (db/m lean,n=10-12). * P<0.05, PEG-Compound 2 (n=13-14) versus Vehicle. ANOVA withDunnett's post-hoc test for each pair at individual time points. Dataexpressed as Mean±S.E.M.

FIG. 69. Cardiac hydroxyproline (HP) content is shown at week 3 oftreatment with isoproterenol and PEG-Compound 2. Each data pointrepresents an individual mouse whole heart HP content normalized toprotein. *: P<0.05, vs Sham+Vehicle, **: P<0.05, vs Iso+Vehicle (One-wayANOVA followed by Bonferroni's test).

FIG. 70A-C. Effects of FGF-21 variants on serum total adiponectin (FIG.70A), serum high molecular weight (HMW) adiponectin (FIG. 70B), and theratio of HMW adiponectin to total adiponectin (FIG. 70C) in a StelicNASH mouse study. All values are mean±SEM.

FIG. 71A-B. Liver weight (FIG. 71A) and liver weight to body weight (BW)ratio (FIG. 71B) in a NASH animal model.

FIG. 72. Effects of FGF-21 variants in a NASH animal model. Followingtreatment with PEGylated Compound 1 (“PEG-Cmpd 1”), PEGylatd Compound 2(“PEG-Cmpd 2”) or Compound 170 (“Cmpd 170”), animals were assessed forbody weight (FIG. 72A), liver weight (FIG. 72B), liver-to-body weightratio (FIG. 72C), liver triglyceride (FIG. 72D), plasma ALT (FIG. 72E),liver cholesterol (FIG. 72F), and plasma triglycerides (FIG. 72G).Statistically significant differences from vehicle-treated controls areas shown. n.s.: no statistically significant difference detected (i.e.,P≧0.05).

DETAILED DESCRIPTION Definitions

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “FGF-21,” “FGF-21polypeptide,” or “modified FGF-21 polypeptide” is a reference to one ormore such proteins and includes equivalents thereof known to those ofordinary skill in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

Normoglycemia: In the present disclosure, the terms normoglycemia oreuglycemia refer to the state of having a normal blood glucoseconcentration. An exemplary normal blood glucose concentration in humansis between 70 mg/dl and 99 mg/dl in fasting adults, and between 70 mg/dland 140 mg/dl in postprandial adults. Sustained normoglycemia refers tomaintenance of normoglycemia for an extensive period of time, e.g., atleast one day, at least two days, at least one week, at least two weeks,at least one month, or longer, for example during ongoing treatment witha modified FGF-21 polypeptide of the present disclosure.

The term “half-life extending moiety” (also referred to herein as“HLEM”) refers to a pharmaceutically acceptable moiety, domain, ormolecule covalently linked (“conjugated” or “fused”) to the modifiedFGF-21 polypeptide described herein, optionally via a non-naturallyencoded amino acid, directly or via a linker, that prevents or mitigatesin vivo proteolytic degradation or other activity-diminishing chemicalmodification of the modified FGF-21 polypeptide, increases half-life,and/or improves or alters other pharmacokinetic or biophysicalproperties including but not limited to increasing the rate ofabsorption, reducing toxicity, improving solubility, reducing proteinaggregation, increasing biological activity and/or target selectivity ofthe modified FGF-21 polypeptide, increasing manufacturability, and/orreducing immunogenicity of the modified FGF-21 polypeptide, compared toa comparator such as an unconjugated form of the modified FGF-21polypeptide or wildtype FGF-21 polypeptide. The term “half-lifeextending moiety” includes non-proteinaceous, half-life extendingmoieties, such as a water soluble polymer such as polyethylene glycol(PEG) or discrete PEG, hydroxyethyl starch (HES), a lipid, a branched orunbranched acyl group, a branched or unbranched C8-C30 acyl group, abranched or unbranched alkyl group, and a branched or unbranched C8-C30alkyl group; and proteinaceous half-life extending moieties, such asserum albumin, transferrin, adnectins (e.g., albumin-binding orpharmacokinetics extending (PIKE) adnectins), Fc domain, andunstructured polypeptide, such as XTEN and PAS polypeptide (e.g.conformationally disordered polypeptide sequences composed of the aminoacids Pro, Ala, and/or Ser), and a fragment of any of the foregoing. Anexamination of the crystal structure of FGF-21 or FGF family member(s)and its interaction with the FGF receptor can indicate which certainamino acid residues have side chains that are fully or partiallyaccessible to solvent. The side chain of a non-naturally encoded aminoacid at these positions may point away from the protein surface and outinto the solvent and thus be linked to, e.g., a water soluble polymer.

The term “albumin binding moiety” as used herein refers to any chemicalgroup capable of binding to albumin, i.e. has albumin binding affinity,for example, albumin-binding or PKE adnectin.

“Albumin binding affinity” may be determined by several methods knownwithin the art. In one method the compound to be measured isradiolabeled with e.g. 125I or 3H and incubated with immobilized albumin(Kurtzhals et. al., Biochem. J., 312, 725-731 (1995)). The binding ofthe compound relative to a standard is calculated. In another method arelated compound is radiolabeled and its binding to albumin immobilizedon e.g. SPA beads (scintillation proximity assay beads, PerkinElmer catno. RPNQ0001) is competed by a dilution series of the compound to bemeasured. The EC50 value for the competition is a measure of theaffinity of the compound. In a third method, the receptor affinity orpotency of a compound is measured at different concentrations ofalbumin, and the shift in relative affinity or potency of the compoundas a function of albumin concentration reflects its affinity foralbumin.

The term “thermal stability” refers to the ability of a polypeptide toresist unfolding when heated. Generally the higher the thermal stabilityof a molecule, the greater the temperature that is required for thepolypeptide to become unfolded. Exemplary methods of determining thethermal stability of a polypeptide are the differential scanningcalorimetry (DSC) and thermal scanning fluorescence methods described inExample 6 herein. Thermal stability may be determined with respect to acomparator compound, e.g., to identify a polypeptide having increasedthermal stability.

The term “aggregation” refers to the tendency of a polypeptide to formnon-covalently linked complexes with other molecules (such as othermolecules of the same polypeptide) thereby forming high molecular weightcomplexes. Exemplary methods of measuring the formation of aggregatesinclude analytical size exclusion chromatography as described in Example7 herein. Relative amounts of aggregation may be determined with respectto a comparator compound, e.g., to identify a polypeptide having reducedaggregation.

The term “deamidation” refers to the tendency of amino acid residueswithin a polypeptide to spontaneously undergo a deamidation reaction,thereby changing the chemical structure of the amino acid, andpotentially affecting the function of the polypeptide. Exemplary methodsof measuring deamidation include imaged capillary isoelectric focusing(icIEF) as described in Example 7 herein. The relative amount ofdeamidation may be determined with respect to a comparator compound,e.g., to identify a polypeptide having decreased deamidation.

The term “in vivo proteolysis” refers to the cleavage of a polypeptidewhen introduced into a living system (e.g., when injected into anorganism) which may result from proteases occurring in said organism.Proteolysis can potentially affect the biological activity or half-lifeof a polypeptide. For example, wild-type FGF-21 can undergo cleavage atthe C-terminus, resulting in a truncated, inactive polypeptide. Anexemplary method of measuring in vivo proteolysis of FGF-21 is the MesoScale Discovery (MSD)-based electrochemiluminescent immunosorbent assay(ECLIA) described in Example 10 herein. The relative amount of in vivoproteolysis may be determined with respect to a comparator compound,e.g., to identify a polypeptide having decreased in vivo proteolysis.

The term “solubility” refers to the amount of a substance that candissolve in another substance, e.g., the amount of an unmodified ormodified FGF-21 polypeptide that can dissolve in an aqueous solution. Anexemplary method of measuring the solubility of an unmodified ormodified FGF-21 polypeptide is the plug flow solubility test describedin Example 8 herein. Relative solubility may be determined with respectto a comparator compound, e.g., to identify a polypeptide havingincreased solubility.

The term “biological activity” refers to the ability of a molecule toaffect any physical or biochemical properties of a biological system,pathway, molecule, or interaction relating to an organism, including butnot limited to, viruses, bacteria, bacteriophage, transposon, prion,insects, fungi, plants, animals, and humans. For example, in the contextof an unmodified or modified FGF-21, biological activity includes any ofthe functions performed by FGF-21 as described herein. Exemplary methodsof determining whether a molecule possesses at least one biologicalactivity of wild-type FGF-21 (such as the wild-type FGF-21 polypeptideof SEQ ID NO: 1) may include the in vitro assay described in Example 5or the in vitro assay described in Example 17. The relative level ofbiological activity may be determined with respect to a comparatorcompound, e.g., to identify a polypeptide having biological activity orhaving sufficiently high biological activity for an intended therapeuticuse, e.g., having an EC50 less than 5-fold, 10-fold, less than 20-fold,less than 50-fold, or less than 100-fold higher than the EC50 of acomparator.

The comparator compound described herein may be another sequence lackinga modification, such as a modification described herein. For example,the comparator compound may be the same modified FGF-21 polypeptidesequence without an internal deletion, without a replacement peptide, orwithout a fusion partner. Exemplary comparator compounds include withoutlimitation the wild-type FGF-21 polypeptide of SEQ ID NO:1, a modifiedFGF-21 polypeptide of SEQ ID NO:201, or another comparator compound. Insome embodiments, the comparator compound may contain at least onenon-naturally encoded amino acid, which may be linked to a linker,polymer, biologically active molecule, peptide, polypeptide, orhalf-life extending moiety described herein (e.g. PEG). In someembodiments, a comparator compound may contain at least onenon-naturally encoded amino acid, which may not be linked to a linker,polymer, biologically active molecule, peptide, polypeptide, orhalf-life extending moiety described herein (e.g. PEG). In someembodiments, a comparator compound may contain additional amino acidsubstitutions, deletions, and/or insertions. In some embodiments, thecomparison may be performed with a pegylated or non-pegylated form ofthe polypeptide; in the former instance, the comparison may be performedwith a polypeptide comprising or not comprising a non-naturally encodedamino acid. In some embodiments, a comparator compound may contain aninternal deletion that is optionally replaced by a peptide (e.g., thesame internal deletion and replacement peptide in the compound to whichthe comparator is being compared), but without a fusion partner.

The term “Fc,” “Fc domain,” or “Fc region” refers to an Fc domain orfragment thereof. An Fc may be a native Fc region comprising an aminoacid sequence identical to the amino acid sequence of an Fc region foundin nature, or a variant Fc region comprising an amino acid sequencewhich differs from that of a native Fc region by virtue of at least oneamino acid. In some embodiments, the Fc has at least one amino acidsubstitution compared to a native Fc region or to the Fc region of aparent polypeptide, e.g., from about one to about twenty, from about oneto about ten, or from about one to about five amino acid substitutions,deletions or insertions in a native Fc region or in the Fc region of theparent polypeptide. The Fc region herein may possess at least about 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a native Fcregion and/or with an Fc region of a parent polypeptide. In someembodiments, the Fc region may have at least about 90% sequence identitywith a native Fc region and/or with an Fc region of a parentpolypeptide. In some embodiments, the Fc region may have at least about95% sequence identity with a native Fc region and/or with an Fc regionof a parent polypeptide. Exemplary amino acid sequences of Fc regionsinclude SEQ ID NOs: 302 and 323-335. Exemplary domains or fragments ofFc regions include the polypeptides of SEQ ID NOs:303-309.

As used herein, a “functional Fc region” refers to an Fc domain orfragment thereof which retains the ability to bind FcRn.

The term “corresponding” refers to a position (“position corresponding”or “corresponding position”) or region (“region corresponding” or“corresponding region”) within a polypeptide or polynucleotide sequencethat is identified by comparison to a reference sequence. The referencesequence may be a wild-type or unmodified sequence, such as thewild-type FGF-21 polypeptide of SEQ ID NO:1. A corresponding position orregion may be identified by alignment of the sequence with a referencesequence. For example, the “position corresponding to amino acid 108 inSEQ ID NO:1” in a sequence refers to the position in the sequence thatis in the same alignment column as amino acid 108 in SEQ ID NO:1 whenthat sequence is aligned with SEQ ID NO:1. In the alignment, the aminoacid or nucleotide may or may not match the amino acid or nucleotide inthe corresponding position in the reference sequence. When referring toa deletion of a corresponding region, the alignment may contain gaps inthe alignment columns corresponding to each position within the deletedregion, unless the deleted region has been replaced by a replacementpeptide which may potentially align with part of the deleted region.Thus, for a deletion replaced by a replacement peptide, the replacementpeptide may be omitted from the sequence when performing the alignment,such that the alignment should contain gaps throughout the deletedregion. Alternatively, the replacement peptide, if present, may bedisregarded when identifying a corresponding region.

The alignment used to identify a corresponding position or correspondingregion may be obtained using a conventional alignment algorithm such asBlast (Altschul et al., J Mol Biol. 1990 Oct. 5; 215(3):403-10),Smith-Waterman (Smith and Waterman, J Mol Biol. 1981 Mar. 25;147(1):195-7), or Needleman-Wunsch (Needleman and Wunsch, J Mol Biol.1970 March; 48(3):443-53). The Needleman-Wunsch algorithm may be used inorder to obtain the highest-scoring global alignment (i.e., an alignmentcontaining every residue in both sequences, though an alignment maystart and/or end in gaps). Whether Blast, Smith-Waterman, orNeedleman-Wunsch is utilized, the highest scoring alignment may beidentified using “default” parameters, such as use of the BLOSUM62scoring matrix, a gap open penalty of 11, and a gap extend penalty of 1,and (when using Blast for pairwise alignment) a word size of 3.

“Region” refers to a contiguous portion of a polypeptide orpolynucleotide sequence. A region may be identified by two positionswithin a polypeptide or polynucleotide that specify the start and endpositions of the region within the sequence. Unless specified otherwise,a region is inclusive of the positions defining the region within thereference sequence, i.e., includes the given start and end positions.For example, the region 119-130 of SEQ ID NO:1 refers to the portion ofSEQ ID NO:1 starting at amino acid 119 and ending at amino acid 130,including amino acids 119 and 130, which has the sequence PGNKSPHRDPAP(SEQ ID NO:73).

The term “deletion” refers to the removal of one or more (or a specifiednumber of) contiguous amino acids or nucleotides from a polypeptide orpolynucleotide. An “internal deletion” refers to a deletion that doesnot include the N- or C-terminus of a polypeptide or the 5′ or 3′ end ofa polynucleotide. A deletion or an internal deletion can be identifiedby specifying the start and end positions of the deletion relative to areference sequence. With respect to a modified FGF-21 polypeptide, areference to an internal deletion generally specifies the deletion of aregion relative to a reference FGF-21 polypeptide, such as a deletion ofa region corresponding to positions of the wild-type FGF-21 polypeptideof SEQ ID NO:1, for example, amino acid positions 119-130 in SEQ ID NO:1(i.e., PGNKSPHRDPAP (SEQ ID NO:73)).

“Replacement peptide” refers to amino acids that are inserted in placeof an internal deletion or other deletion within a polypeptide. Thelength of the replacement peptide may differ from the length of theinternal deletion. Exemplary replacement peptides may comprise one ormore glycine, serine, and histidine residues, and in some instances,additional amino acid residues such as lysine and proline. However, itis to be understood that a replacement peptide is not limited tosequences containing these particular amino acids, but rather mayinclude any natural amino acid or non-naturally encoded amino acid. Areplacement peptide may be of any length, e.g., a single amino acid(i.e., having a length of 1 amino acid), two or more amino acids, orthree or more amino acids. A replacement peptide having a length of zeroamino acids indicates that a replacement peptide is not present. In someembodiments, the replacement polypeptide may have a length of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids. In some embodiments, thereplacement polypeptide may have a length of 1-10, 1-7, 1-5, or 1-3amino acids. In some embodiments, the replacement polypeptide may have alength of 2-7, 2-5 or 2-3 amino acids. In some embodiments, thereplacement polypeptide may have a length of 3 amino acids. Exemplaryreplacement peptides include the sequences G, GG, SG, GSG, GGH, SGG,GSGH (SEQ ID NO:343), HSG, HHSG (SEQ ID NO:344), HGSH (SEQ ID NO:345),GSGP (SEQ ID NO:346), HGG, HSGG (SEQ ID NO:347), and HGSG (SEQ IDNO:348). Additional exemplary replacement peptides include the sequencesK, DKS, HKS, D, Q, KDS, and KDSQ (SEQ ID NO:349).

“Fusion partner” refers to a peptide or polypeptide fused to themodified FGF-21 polypeptide described herein. The fusion partner may befused to the modified FGF-21 polypeptide on the N- and/or C-terminus.Exemplary fusion partners include, but are not limited to, albumin,transferrin, adnectins (e.g., albumin-binding or pharmacokineticsextending (PIKE) adnectins), Fc domain, and unstructured polypeptide,such as XTEN and PAS polypeptide (e.g. conformationally disorderedpolypeptide sequences composed of the amino acids Pro, Ala, and/or Ser),or a fragment of any of the foregoing. The fusion partner may be fusedto the modified FGF-21 polypeptide for any purpose, including but notlimited to, purification, manufacturability, half-life extension,enhanced biophysical properties (e.g. solubility or stability), reducedimmunogenicity or toxicity, etc.

“Connecting peptide” refers to an amino acid sequence having both its N-and C-termini fused to other peptides or polypeptides. A connectingpeptide may be present in a fusion protein, e.g., having its terminifused (in either order) to a modified FGF-21 polypeptide and a fusionpartner. Exemplary connecting peptides may comprise between 0 aminoacids (i.e., no connecting peptide present) and 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,60, or 100 amino acids, or more. In some embodiments, the connectingpeptide may comprise between 1 and 40, between 1 and 30, between 1 and20, between 1 and 10, between 1 and 5, between 2 and 40, between 2 and30, between 2 and 20, between 2 and 10, between 5 and 40, between 5 and30, between 5 and 20, or between 5 and 10 amino acids. In someembodiments, the connecting peptide may comprise between 5 and 20 aminoacids. In some embodiments, the connecting peptide may comprise between10 and 20 amino acids. Certain exemplary connecting peptides describedherein may be rich in serine and glycine residues, however, it is to beunderstood that the connecting peptide is not limited to such sequences.Exemplary connecting peptides include the following sequences: GAGGGGSG(SEQ ID NO:74), EPKSSD (SEQ ID NO:75), D, ESPKAQASSVPTAQPQAEGLA (SEQ IDNO:76), ELQLEESAAEAQDGELD (SEQ ID NO:77), GQPDEPGGS (SEQ ID NO:78),GGSGSGSGSGSGS (SEQ ID NO:79), ELQLEESAAEAQEGELE (SEQ ID NO:80), GSGSG(SEQ ID NO:81), GSGC (SEQ ID NO:82), AGGGGSG (SEQ ID NO:83), GSGS (SEQID NO:84), QPDEPGGS (SEQ ID NO:85), GSGSGS (SEQ ID NO:86), TVAAPS (SEQID NO:87), KAGGGGSG (SEQ ID NO:88), KGSGSGSGSGSGS (SEQ ID NO:89),KQPDEPGGS (SEQ ID NO:90), KELQLEESAAEAQDGELD (SEQ ID NO:91), KTVAAPS(SEQ ID NO:92), KAGGGGSGG (SEQ ID NO:93), KGSGSGSGSGSGSG (SEQ ID NO:94),KQPDEPGGSG (SEQ ID NO:95), KELQLEESAAEAQDGELDG (SEQ ID NO:96), KTVAAPSGAGGGGSGG (SEQ ID NO:97), AGGGGSG (SEQ ID NO:98), GSGSGSGSGSGSG (SEQ IDNO:99), QPDEPGGSG (SEQ ID NO:100), TVAAPSG (SEQ ID NO:301),GGGGSGGGSGGGGGSGGGSGGGGSGGGS (SEQ ID NO:350), PSPEPPTPEPPSPEP (SEQ IDNO:351), ELQLEESAAEAQEGELE (SEQ ID NO:352), SSGGGGSGGGSGGGGGS (SEQ IDNO:353), GS (SEQ ID NO: 354), GGGGS (SEQ ID NO: 355), EEEEDEEEED (SEQ IDNO: 356), PSPEPPTPEP (SEQ ID NO: 357), GSHHHHHHHHGS (SEQ ID NO: 358),GGGGSGGGGSGGGGS (SEQ ID NO: 359), GGGGGSGGGSGGGGS (SEQ ID NO: 360),GSGSGSGSGSGSGSGS (SEQ ID NO: 361), PSTPPTPSPSTPPTPSPS (SEQ ID NO: 362),RGGEEKKKEKEKEEQEERETKTP (SEQ ID NO: 363), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQID NO: 364), PSPEPPTPEPPSPEPPTPEPPSPEPPTPEP (SEQ ID NO: 365),PSTPPTPSPSTPPTPSPSPSTPPTPSPSTPPTPSPS (SEQ ID NO: 366), PSPEP (SEQ ID NO:367), PSPEPPTPEPPSPEPPTPEP (SEQ ID NO: 368),PSPEPPTPEPPSPEPPTPEPPSPEPPTPEPPSPEPPTPEP (SEQ ID NO: 369),PTPEPPSPEPPTPEPPSPEP (SEQ ID NO: 370), PSPEPGGGSPTPEP (SEQ ID NO: 371),PSPEPEEEDPTPEP (SEQ ID NO: 372), PSPEPPTPEPEEEDPSPEPPTPEP (SEQ ID NO:373), PTPEPPSPEPPTPEPEEEDPSPEPPTPEPPSPEP (SEQ ID NO: 374),PTPEPPSPEPPTPEPGGGGSPSPEPPTPEPPSPEP (SEQ ID NO: 375),PSPEPTPEPSPEPPTPEPSPEPTPEP (SEQ ID NO: 376), GETGS (SEQ ID NO: 377),GGGGSGGGGS (SEQ ID NO: 378), GETGSSGEGT (SEQ ID NO: 379),GETGSSGEGTGSTGS (SEQ ID NO: 380), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 381),GETGSSGEGTGSTGSGAGES (SEQ ID NO: 382), andGETGSSGEGTGSTGSGAGESGTGESGEGGS (SEQ ID NO: 383), i.e., SEQ IDNOs:74-100, 301, and 350-383.

The term “disease associated with fibrosis” includes diseases,disorders, and conditions in which fibrosis has been observed to occuror in which fibrosis is known or thought to be associated with orcontribute to disease etiology, progression, or symptoms, or in whichfibrosis is known or thought to occur as the disease progresses. Thefibrosis may affect an organ or tissue such as the pancreas, lung,heart, kidney, liver, eyes, nervous system, bone marrow, lymph nodes,endomyocardium, or retroperitoneum. Exemplary diseases associated withfibrosis include, but are not limited to nonalcoholic steatohepatitis(NASH), liver fibrosis, pre-cirrhosis, cirrhosis, diffuse parenchymallung disease, cystic fibrosis, lung or pulmonary fibrosis, progressivemassive fibrosis, idiopathic pulmonary fibrosis, injection fibrosis,kidney or renal fibrosis, chronic kidney disease, diabetic kidneydisease, focal segmental glomerulosclerosis, membranous nephropathy, IgAnephropathy, myelofibrosis, heart failure, metabolic heart failure,cardiac fibrosis, cataract fibrosis, cataract, ocular scarring,pancreatic fibrosis, skin fibrosis, intestinal fibrosis, intestinalstrictures, endomyocardial fibrosis, atrial fibrosis, mediastinalfibrosis, Crohn's disease, retroperitoneal fibrosis, keloid, nephrogenicsystemic fibrosis, scleroderma, systemic sclerosis, arthrofibrosis,Peyronie's syndrome, Dupuytren's contracture, diabetic neuropathy,adhesive capsulitis, alcoholic liver disease, hepatosteatosis, viralhepatitis, biliary disease, primary hemochromatosis, drug-relatedcirrhosis, cryptogenic cirrhosis, Wilson's disease, and, alpha1-antitrypsin deficiency, interstitial lung disease (ILD), humanfibrotic lung disease, macular degeneration, retinal retinopathy,vitreal retinopathy, myocardial fibrosis, Grave's ophthalmopathy, druginduced ergotism, cardiovascular disease, atherosclerosis/restenosis,hypertrophic scars, primary or idiopathic myelofibrosis, andinflammatory bowel disease (including, but not limited to, collagenouscolitis). In some embodiments, the disease associated with fibrosis mayinclude liver fibrosis, kidney or renal fibrosis, lung or pulmonaryfibrosis and heart or cardiac fibrosis. In some embodiments, the diseaseassociated with fibrosis may be liver fibrosis. In some embodiments, thedisease associated with fibrosis may be NASH.

The phrase “medically complicated obesity” (also sometimes referred toas “morbid obesity”) generally refers to the condition of a subset ofobese individuals who also have health complications related to orcaused by obesity. Obesity may be determined by determining body massindex (weight in kilograms divided by the square of the height inmeters), with a body mass index of 30 m/kg² or greater indicatingobesity. Exemplary health complications present in medically complicatedobesity may include one or more of type 2 diabetes mellitus,hypertension, obstructive sleep apnea, coronary artery disease and othercardiovascular disease (including coronary artery disease, stroke, andcongestive heart failure), and dyslipidemia. Additional healthcomplications that may be present include nonalcoholic fatty liverdisease (such as steatosis, steatohepatitis, or cirrhosis), respiratorydisease, obstructive sleep apnea, obesity-hypoventilation syndrome,asthma, restrictive lung disease, cancers, osteoarthritis,cholelithiasis, gastroesophageal reflux disease, gynecologicabnormalities, infertility, abnormal menses, venous stasis, skinproblems, intertrigo, cellulitis, increased risk of complications duringsurgery or pregnancy, urinary incontinence and idiopathic intracranialhypertension. Medically complicated obesity may also be associated withPrader-Willi Syndrome.

The term “substantially purified” refers to an unmodified or modifiedFGF-21 polypeptide that may be substantially or essentially free ofcomponents that normally accompany or interact with the protein as foundin its naturally occurring environment, i.e. a native cell, or host cellin the case of recombinantly produced unmodified or modified FGF-21polypeptides. Unmodified or modified FGF-21 polypeptide that may besubstantially free of cellular material includes preparations of proteinhaving less than about 30%, less than about 25%, less than about 20%,less than about 15%, less than about 10%, less than about 5%, less thanabout 4%, less than about 3%, less than about 2%, or less than about 1%(by dry weight) of contaminating protein. When the unmodified ormodified FGF-21 polypeptide or variant thereof is recombinantly producedby the host cells, the protein may be present at about 30%, about 25%,about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%,or about 1% or less of the dry weight of the cells. When the unmodifiedor modified FGF-21 polypeptide or variant thereof is recombinantlyproduced by the host cells, the protein may be present in the culturemedium at about 5 g/L, about 4 g/L, about 3 g/L, about 2 g/L, about 1g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about 100 mg/L,about 50 mg/L, about 10 mg/L, or about 1 mg/L or less of the dry weightof the cells. Thus, “substantially purified” unmodified or modifiedFGF-21 polypeptide as produced by the methods of the present disclosuremay have a purity level of at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%,specifically, a purity level of at least about 75%, 80%, 85%, and morespecifically, a purity level of at least about 90%, a purity level of atleast about 95%, a purity level of at least about 99% or greater asdetermined by appropriate methods such as SDS/PAGE analysis, RP-HPLC,SEC, and capillary electrophoresis.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells, prokaryotic host cells, E. coli, orPseudomonas host cells, and cell contents. Thus, the term may encompassmedium in which the host cell has been grown, e.g., medium into whichthe unmodified or modified FGF-21 polypeptide has been secreted,including medium either before or after a proliferation step. The termalso may encompass buffers or reagents that contain host cell lysates,such as in the case where the unmodified or modified FGF-21 polypeptideis produced intracellularly and the host cells are lysed or disrupted torelease the unmodified or modified FGF-21 polypeptide.

The term “anti-diabetic agent” shall mean any drug that is useful intreating, preventing, or otherwise reducing the severity of any glucosemetabolism disorder, or any complications thereof, including any of theconditions, disease, or complications described herein. Anti-diabeticagents include insulin, thiazolidinediones, sulfonylureas, benzoic acidderivatives, alpha-glucosidase inhibitors, or the like. The inventiveantidiabetic compositions may be capable of reducing HbA1c levels by atleast a 10% or at least a 50% change from the baseline. Antidiabeticagents include insulin potentiators, such as including but not limitedto, small molecule insulin potentiators, Taurine, Alpha Lipoic Acid, anextract of Mulberry, Chromium, Glutamine, Enicostemma littorale Blume,Scoparia dulcis, an extract of Tarragon, Andrographis paniculata,Isomalt, Trehalose or D-Mannose which may further potentiate thesecretion or activity of insulin.

As used herein, “modified FGF-21 polypeptide,” “modified fibroblastgrowth factor 21” or “modified FGF-21” and unhyphenated forms thereofare used interchangeably and shall include those polypeptides andproteins that differ from wild-type FGF-21 (e.g., wild-type human FGF-21of SEQ ID NO:1 and SEQ ID NO:5) and typically have at least onebiological activity of a fibroblast growth factor 21, as well as FGF-21analogs, FGF-21 isoforms, FGF-21 mimetics, FGF-21 fragments, hybridFGF-21 proteins, fusion proteins, oligomers and multimers, homologues,glycosylation pattern variants, variants, splice variants, and muteins,thereof, regardless of the biological activity of same. The term“modified FGF-21 polypeptide” and “modified FGF-21” encompass FGF-21polypeptides comprising one or more amino acid substitutions, additionsor deletions. For example, modified FGF-21 polypeptides of the presentdisclosure comprise an internal deletion, including those internaldeletions shown in FIG. 1 and others described herein.

The term “modified FGF-21 polypeptide” also encompasses polymorphisms(e.g., naturally occurring FGF-21 sequence variants). For example, the“P-form” of FGF-21 contains a proline (P) at position 174 (position 146in the mature polypeptide of SEQ ID NO:1), while the “L-form” contains aleucine (L) at position 174 (position 146 in the mature polypeptide ofSEQ ID NO:5). Exemplary P-form FGF-21 polypeptide sequences arecontained in SEQ ID NOs: 1-4 while exemplary L-form FGF-21 polypeptidesequences are contained in SEQ ID NOs: 5-7.

Substitutions in a wide variety of amino acid positions innaturally-occurring FGF-21 have been described. Substitutions includingbut not limited to, those that modulate solubility or stability,increase agonist activity, increase in vivo or in vitro half-life,increase protease resistance, convert the polypeptide into anantagonist, reduce immunogenicity or toxity, facilitate purification ormanufacturability, etc. and are encompassed by the term “modified FGF-21polypeptide” or “modified FGF-21.” In some cases, the non-naturallyencoded amino acid substitution(s) may be combined with other additions,substitutions or deletions within the modified FGF-21 polypeptide toaffect other biological traits of the modified FGF-21 polypeptiderelative to another FGF-21 polypeptide (e.g., the wild-type FGF-21polypeptide of SEQ ID NO:1, the modified FGF-21 polypeptide of SEQ IDNO:201, or another FGF-21 polypeptide such as the same FGF-21polypeptide without said addition, substitution, or deletion, or anotherunmodified or modified FGF-21 unmodified or modified polypeptide). Insome cases, the other additions, substitutions or deletions may increasethe stability (including but not limited to, resistance to proteolyticdegradation) of the modified FGF-21 polypeptide or increase affinity ofthe modified FGF-21 polypeptide for its receptor. In some cases, theother additions, substitutions or deletions may increase thepharmaceutical stability of the modified FGF-21 polypeptide. In somecases, the other additions, substitutions or deletions may increase thesolubility (including but not limited to, when expressed in E. coli orother host cells) of the modified FGF-21 polypeptide. In someembodiments the additions, substitutions or deletions may increase thepolypeptide solubility following expression in E. coli or otherrecombinant host cells. In some embodiments sites are selected forsubstitution with a naturally encoded or non-natural amino acid inaddition to another site for incorporation of a non-natural amino acidthat results in increasing the polypeptide solubility followingexpression in E. coli or other recombinant host cells. In someembodiments, the modified FGF-21 polypeptides comprise another addition,substitution or deletion that modulates affinity for the FGF-21polypeptide receptor, binding proteins, or associated ligand, modulatessignal transduction after binding to the FGF-21 receptor, modulatescirculating half-life, modulates release or bio-availability,facilitates purification, or improves or alters a particular route ofadministration. In some embodiments, the modified FGF-21 polypeptidescomprise an addition, substitution or deletion that increases theaffinity of the modified FGF-21 for its receptor. Similarly, modifiedFGF-21 polypeptides can comprise chemical or enzyme cleavage sequences,protease cleavage sequences, reactive groups, antibody-binding domains(including but not limited to, FLAG or poly-His) or other affinity basedsequences (including, but not limited to, FLAG, poly-His, GST, etc.) orlinked molecules (including, but not limited to, biotin) that improvedetection (including, but not limited to, GFP), purification, transportthrough tissues or cell membranes, prodrug release or activation,modified FGF-21 size reduction, or other traits of the polypeptide.

For sequences of FGF-21 that lack a leader sequence, see SEQ ID NO: 1,SEQ ID NO: 2 and SEQ ID NO: 5 herein. For sequences of FGF-21 with aleader sequence, see SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ IDNO: 7 herein. In some embodiments, FGF-21 polypeptides of the disclosureare substantially identical to SEQ ID NOs: 1-7 or any other sequence ofa FGF-21 polypeptide. Multiple polymorphisms of FGF-21 have beenidentified. Leucine or proline have been described at the same positionin U.S. Patent Publication No. 20010012628 and U.S. Pat. No. 6,716,626.N-terminal leader or signal sequences that differ by 1 amino acid(leucine) are shown in U.S. Pat. No. 6,716,626 and U.S. PatentPublication No. 20040259780. FGF-21 polypeptide variants or mutantsinclude, but are not limited to, those disclosed in U.S. Pat. No.6,716,626; U.S. Patent Publication Nos. 2005/0176631, 2005/0037457,2004/0185494, 2004/0259780, 2002/0164713, and 2001/0012628; WO 01/36640;WO 03/011213; WO 03/059270; WO 04/110472; WO 05/061712; WO 05/072769; WO05/091944; WO 05/113606; WO 06/028595; WO 06/028714; WO 06/050247; WO06/065582; WO 06/078463; WO01/018172; WO09/149171; WO10/042747;WO12/066075; WO11/154349; WO13/052311; WO13/188181, which areincorporated by reference in their entirety herein.

The term “modified FGF-21 polypeptide” also includes biologically-activefragments, biologically active variants and stereoisomers of thenaturally-occurring FGF-21 as well as agonist, mimetic, and antagonistvariants of the naturally-occurring FGF-21 and polypeptide fusionsthereof. Fusions comprising additional amino acids at the aminoterminus, carboxyl terminus, or both, are encompassed by the term“modified FGF-21 polypeptide.” Exemplary fusions include, but are notlimited to, e.g., methionyl FGF-21 in which a methionine is linked tothe N-terminus of FGF-21 resulting from the recombinant expression ofthe mature form of FGF-21 lacking the leader or signal peptide orportion thereof (a methionine is linked to the N-terminus of FGF-21resulting from the recombinant expression, e.g. in E. coli), fusions forthe purpose of purification (including, but not limited to, topoly-histidine or affinity epitopes), fusions with serum albumin bindingpeptides such as PKE adnectin and fusions with serum proteins such asserum albumin, and fusion proteins comprising FGF-21 and one or moreother molecules (“fusion partner”), including but not limited to, serumalbumin, Fc domain, immunoglobulin constant region, unstructuredpolypeptide, and adnectin, and a fragment thereof. Any such fragmentscan be prepared from the proteins by standard biochemical methods, or byexpressing a polynucleotide encoding the fragment.

Except where indicated otherwise, in general the terms “FGF-21polypeptide” “fibroblast growth factor 21” and “FGF-21” as used hereinencompasses both unmodified (i.e., wild-type) FGF-21 and modified FGF-21polypeptides.

The term “modified FGF-21 polypeptide” includes polypeptides conjugatedto a polymer such as PEG and may optionally comprise one or moreadditional derivitizations of cysteine, lysine, or other residues. Inaddition, the modified FGF-21 polypeptide may comprise a linker orpolymer, wherein the amino acid to which the linker or polymer isconjugated may be a non-natural amino acid according to the presentdisclosure, or may be conjugated to a naturally encoded amino acidutilizing techniques known in the art such as coupling to lysine orcysteine.

The term “modified FGF-21 polypeptide” also includes glycosylatedmodified FGF-21, such as but not limited to, polypeptides glycosylatedat any amino acid position, N-linked or O-linked glycosylated forms ofthe polypeptide. Variants containing single nucleotide changes are alsoconsidered as biologically active variants of FGF-21 polypeptide. Inaddition, splice variants are also included. The term “modified FGF-21polypeptide” also includes FGF-21 polypeptide heterodimers, homodimers,heteromultimers, or homomultimers of any one or more unmodified ormodified FGF-21 polypeptides or any other polypeptide, protein,carbohydrate, polymer, small molecule, linker, ligand, or otherbiologically active molecule of any type, linked by chemical means orexpressed as a fusion protein, as well as polypeptide analoguescontaining, for example, specific deletions or other modifications yetmaintain biological activity.

All references to amino acid positions in unmodified or modified FGF-21described herein are based on the corresponding position in SEQ ID NO:1, unless otherwise specified (i.e., when it is stated that thecomparison is based on SEQ ID NO: 2, 3, 4, 5, 6, 7, or other FGF-21sequence). For example, the amino acid at position 77 of SEQ ID NO: 1,is an arginine and the corresponding arginine is located in SEQ ID NO: 2at position 87. Those of skill in the art will appreciate that aminoacid positions corresponding to positions in SEQ ID NO: 1 can be readilyidentified in any other FGF-21 molecule such as SEQ ID NO: 2, 3, 4, 5,6, and 7. Those of skill in the art will appreciate that amino acidpositions corresponding to positions in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7or any other FGF-21 sequence can be readily identified in any otherFGF-21 molecule such as FGF-21 fusions, variants, fragments, etc. Forexample, sequence alignment programs such as BLAST can be used to alignand identify a particular position in a protein that corresponds with aposition in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or other FGF-21 sequence.Substitutions, deletions or additions of amino acids described herein inreference to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or other FGF-21 sequenceare intended to also refer to substitutions, deletions or additions incorresponding positions in FGF-21 fusions, variants, fragments, etc.described herein or known in the art and are expressly encompassed bythe present disclosure.

The term “modified FGF-21 polypeptide” or “modified FGF-21” encompassesFGF-21 polypeptides comprising one or more amino acid substitutions,insertions or deletions. For example, modified FGF-21 polypeptides ofthe present disclosure may be comprised of modifications with one ormore natural amino acids, optionally in conjunction with one or morenon-natural amino acid modification. Exemplary substitutions, insertionsor deletions in a wide variety of amino acid positions in FGF-21polypeptides (including those described herein and others), includingbut not limited to substitutions that modulate pharmaceutical stability,that modulate one or more of the biological activities of the FGF-21polypeptide, such as but not limited to, increase agonist activity,increase solubility of the polypeptide, decrease proteasesusceptibility, decrease deamidation, convert the polypeptide into anantagonist, reduce immunogenicity or toxity, or facilitate purificationor manufacturability, etc. and are encompassed by the term “modifiedFGF-21 polypeptide.”

In some embodiments, the modified FGF-21 polypeptides further comprisean additional insertion, substitution or deletion that modulatesbiological activity of the modified FGF-21 polypeptide. For example, theadditions, substitutions or deletions may modulate one or moreproperties or activities of modified FGF-21. For example, the additions,substitutions or deletions may modulate affinity for the FGF-21polypeptide receptor, modulate circulating half-life, modulatetherapeutic half-life, modulate stability of the polypeptide, modulatecleavage by proteases, modulate dose, modulate release orbio-availability, facilitate purification, decrease deamidation, improveshelf-life, or improve or alter a particular route of administration.Similarly, modified FGF-21 polypeptides may comprise protease cleavagesequences, reactive groups, antibody-binding domains (including but notlimited to, FLAG or poly-His) or other affinity based sequences(including but not limited to, FLAG, poly-His, GST, etc.) or linkedmolecules (including but not limited to, biotin) that improve detection(including but not limited to, GFP), purification or other traits of thepolypeptide.

The term “modified FGF-21 polypeptide” also encompasses homodimers,heterodimers, homomultimers, and heteromultimers that are formed viafusion partners, such as Fc domains, or that are linked, including butnot limited to those linked directly via non-naturally encoded aminoacid side chains, either to the same or different non-naturally encodedamino acid side chains, to naturally-encoded amino acid side chains, orindirectly via a linker. Exemplary linkers including but are not limitedto, small organic compounds, water soluble polymers of a variety oflengths such as poly(ethylene glycol) or polydextran, or polypeptides ofvarious lengths.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally encoded amino acidsinclude, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, Fc domain, immunoglobulinconstant region, unstructured polypeptide, adnectin, or a fragmentthereof, or other moieties that increase serum (in vivo) half-life ofpeptides.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to a living organism.In particular, as used herein, biologically active molecules include,but are not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs,carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides,radionuclides, oligonucleotides, toxoids, toxins, polysaccharides,nucleic acids, peptides, polypeptides, proteins, and portions thereofobtained or derived from viruses, bacteria, insects, animals or anyother cell or cell type, liposomes, microparticles and micelles.

The term “substituents” includes but is not limited to “non-interferingsubstituents”. “Non-interfering substituents” are those groups thatyield stable compounds. Suitable non-interfering substituents orradicals include, but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₁-C₁₂ aralkyl, C₁-C₁₂ alkaryl,C₃-C₁₂ cycloalkyl, C₃-C₁₂ cycloalkenyl, phenyl, substituted phenyl,toluoyl, xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₂-C₁₂ alkoxyaryl,C₇-C₁₂ aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀alkylsulfonyl, —(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8,aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclicradical, substituted heterocyclic radical, nitroalkyl, —NO₂, —CN,—NRC(O)—(C₁-C₁₀ alkyl), —C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkyl thioalkyl,—C(O)O—(C₁-C₁₀ alkyl), —OH, —SO₂, ═S, —COOH, —NR₂, carbonyl,—C(O)—(C₁-C₁₀ alkyl)-CF3, —C(O)—CF3, —C(O)NR2, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀aryl), —C(O)—(C₁-C₁₀ aryl), —(CH₂)_(m)—O—(—(CH₂)_(m)—O—(C₁-C₁₀ alkyl)wherein each m is from 1 to 8, —C(O)NR₂, —C(S)NR₂, SO₂NR₂, —NRC(O) NR₂,—NRC(S) NR₂, salts thereof, and the like. Each R as used herein is H,alkyl or substituted alkyl, aryl or substituted aryl, aralkyl, oralkaryl.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto modified FGF-21 polypeptides can result in changes including, but notlimited to, increased or modulated serum (in vivo) half-life, orincreased or modulated therapeutic half-life relative to the unmodifiedform, modulated immunogenicity or toxicity, modulated physicalassociation characteristics such as aggregation and multimer formation,altered receptor binding, altered binding to one or more bindingpartners, and altered receptor dimerization or multimerization. Thewater soluble polymer may or may not have its own biological activity,and may be utilized as a linker for attaching modified FGF-21 to othersubstances, including but not limited to one or more unmodified ormodified FGF-21 polypeptides, or one or more biologically activemolecules. Suitable polymers include, but are not limited to,polyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C10alkoxy or aryloxy derivatives thereof (described in U.S. Pat. No.5,252,714 which is incorporated by reference herein),monomethoxy-polyethylene glycol, discrete PEG, polyvinyl pyrrolidone,polyvinyl alcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include, butare not limited to, polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” (PEG) or “poly(alkeneglycol)” refers to polyethylene glycol (poly(ethylene glycol)),polypropylene glycol, polybutylene glycol, and derivatives thereof. Theterm “polyalkylene glycol” encompasses both linear and branched polymersand average molecular weights of between 0.1 kDa and 100 kDa. Otherexemplary embodiments are listed, for example, in commercial suppliercatalogs, such as Shearwater Corporation's catalog “Polyethylene Glycoland Derivatives for Biomedical Applications” (2001).

As used herein, the terms “modulated serum half-life” or “modulated invivo half-life” and similar terms refer to the positive or negativechange in circulating half-life of a modified FGF-21 relative to acomparator such as its non-modified form. Serum half-life can bemeasured by taking blood samples at various time points afteradministration of a modified FGF-21, and determining the concentrationof that molecule in each sample. Correlation of the serum concentrationwith time allows calculation of the serum half-life. Increased serum (invivo) half-life desirably may be at least about two-fold, but a smallerincrease may be useful, for example where it enables a satisfactorydosing regimen or avoids a toxic effect. In some embodiments, theincrease may be at least about three-fold, at least about five-fold, orat least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the serum or in vivo half-life of thetherapeutically effective amount of the modified FGF-21 polypeptidedescribed herein, relative to a comparator such as its non-modified formor the wildtype FGF-21. Therapeutic half-life is measured by measuringpharmacokinetic and/or pharmacodynamic properties of the molecule atvarious time points after administration. Increased therapeutichalf-life desirably enables a particular beneficial dosing regimen, aparticular beneficial total dose, or avoids an undesired effect. In someembodiments, the increased therapeutic half-life results from increasedpotency, increased or decreased binding of the modified molecule to itstarget, increased or decreased breakdown of the molecule by enzymes suchas proteases, or an increase or decrease in another parameter ormechanism of action of the non-modified molecule or an increase ordecrease in receptor-mediated clearance of the molecule.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of thecellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to, anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

“Conservatively modified variants” refers to amino acid sequencescontaining conservative substitutions. Exemplary conservatively modifiedvariants include substitutions, deletions or insertions to a nucleicacid, peptide, polypeptide, or protein sequence which alters, adds ordeletes a single amino acid or a small percentage of amino acids in thepolypeptide sequence or encoded polypeptide sequence, e.g., up to 1, 2,3, 4, or 5 amino acids, or up to 0.5%, 1%, 1.5%, 2%, 2.5%, or 3.5% ofthe amino acids in the polypeptide sequence or encoded polypeptidesequence, which optionally may be or may include substitution of aminoacid(s) with chemically similar amino acid(s). Conservative substitutiontables providing functionally similar amino acids are known to those ofordinary skill in the art. Such conservatively modified variants are inaddition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the disclosed modified FGF-21 polypeptides.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, about 96%, about 97%, about 98%, about 99%, identity over aspecified region), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms (or other algorithmsavailable to persons of ordinary skill in the art) or by manualalignment and visual inspection. The identity may exist over a region orcomparison window that is at least about 50 amino acids or nucleotidesin length, or over a region that is 75-100 amino acids or nucleotides inlength, or, where not specified, across the entire sequence of apolynucleotide or polypeptide. A “comparison window”, as used herein,includes reference to a segment of any one of the number of contiguouspositions selected from the group consisting of from 20 to 600, usuallyabout 50 to about 200, more usually about 100 to about 150 in which asequence may be compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.Examples of algorithms that may be suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively, as well as the Smith-Waterman (Smith and Waterman, J MolBiol. 1981 Mar. 25; 147(1):195-7), or Needleman-Wunsch (Needleman andWunsch, J Mol Biol. 1970 March; 48(3):443-53) algorithms, which may berun with the default parameters, e.g., as described in those respectivepublications.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiments a human, who is theobject of treatment, observation or experiment. An animal may be acompanion animal (e.g., dogs, cats, and the like), farm animal (e.g.,cows, sheep, pigs, horses, and the like) or a laboratory animal (e.g.,rats, mice, guinea pigs, and the like).

The term “effective amount” as used herein refers to that amount of thecompound (e.g., a modified FGF-21 polypeptide described herein) beingadministered which may relieve to some extent one or more of thesymptoms of the disease, condition or disorder being treated.Compositions containing the modified FGF-21 polypeptide described hereincan be administered for prophylactic, enhancing, and/or therapeutictreatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

In prophylactic applications, compositions containing the modifiedFGF-21 polypeptide are administered to a patient susceptible to orotherwise at risk of a particular disease, disorder or condition. Suchan amount is defined to be a “prophylactically effective amount.”

In therapeutic applications, compositions comprising the modifiednon-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest or alleviate the symptoms of thedisease, disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and may depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to prophylactic and/or therapeutictreatments.

Non-naturally encoded amino acid polypeptides presented herein mayinclude isotopically-labelled compounds with one or more atoms replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature.

All isomers including but not limited to diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein. In additional or further embodiments, the non-naturally encodedamino acid polypeptides are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In further oradditional embodiments are active metabolites of non-naturally encodedamino acid polypeptides.

I. Overview

Modified FGF-21 molecules comprising an internal deletion and optionallycomprising at least one unnatural amino acid are provided in thedisclosure. Exemplary embodiments of modified FGF-21 polypeptidescomprising an internal deletion are demonstrated to exhibit at least oneadvantageous properties, including, but not limited to, increasedthermal stability, improved solubility, decreased deamination, improvedmanufacturability, improved in vivo half-life of a full-lengthbiologically active form, while retaining a level of biological activitycomparable to an FGF-21 polypeptide without the internal deletion.

Deamidation was mitigated and shelf-life and purity improved in modifiedFGF-21 polypeptides containing a deleted region and optionally replacedwith a peptide (see FIG. 1). Exemplary modified sequences were shown tomitigate a deamidation event that can occur during storage of themolecule. In addition to deamidation mitigation, this modification alsoincreased the thermal stability and/or solubility of the protein insolution and further minimized protein aggregation propensities relativeto wild-type FGF-21 or FGF-21 polypeptides lacking this deletion. Thedecreased deamidation, increased thermal stability, increasedsolubility, and/or decreased aggregation indicate superior formulationcharacteristics, such as longer shelf-life and greater purity, as wellas formulation at greater concentration. Additionally, mitigating thepropensity for deamidation allows a greater range of formulationoptions; otherwise, formulation conditions would need to be selected inorder to decrease deamidation, potentially resulting in otherundesirable formulation characteristics. Furthermore, it isunpredictable and unexpected that a modified FGF-21 polypeptidecomprising an internal deletion of between 5 and 19 contiguous aminoacids and a replacement peptide, may retain the biological function ofFGF-21 and have at least one of decreased deamidation, increased thermalstability, increased solubility, and decreased aggregation.

A further exemplary modification mitigates the in vivo proteolyticclipping of ten C-terminal amino acids from the protein. Thismodification extends the blood half life of the intact, active moleculeresulting in decreases in overall dose, and less frequent dosing. Anexemplary modification that mitigates proteolytic clipping is the pointmutation, G170E, though other modifications may also be utilized.

In some embodiments, the disclosure provides a modified FGF-21polypeptide comprising a polypeptide having an amino acid sequenceselected from SEQ ID NOs: 1-7, except that said amino acid sequence maycomprise: (i) an internal deletion of between 2 and 19 amino acids (suchas between 5 and 19 amino acids), wherein said internal deletion iswithin a region corresponding to amino acids 116 to 134 of SEQ ID NO:1,wherein said internal deletion is replaced by a replacement peptidehaving a length of between 0 and 12 amino acids; and (ii) 9 or feweradditional amino acid substitutions, deletions, and/or insertions.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycontain at least one non-naturally encoded amino acid. In someembodiments, said at least one non-naturally encoded amino acid is at aposition corresponding to amino acid 72, 77, 86, 87, 91, 108, 110, 126,131, or 146 of SEQ ID NO: 1. In some embodiments, said at least onenon-naturally encoded amino acid is at a position corresponding to aminoacid 77, 91, 108 or 131 of SEQ ID NO: 1. In some embodiments, said atleast one non-naturally encoded amino acid is at a positioncorresponding to amino acid 108 in SEQ ID NO: 1. In some embodiments,said at least one non-naturally encoded amino acid is at a positioncorresponding to amino acid 72 in SEQ ID NO: 1. In some embodiments,said at least one non-naturally encoded amino acid is at a positioncorresponding to amino acid 77 in SEQ ID NO: 1. In some embodiments,said at least one non-naturally encoded amino acid is at a positioncorresponding to amino acid 86 in SEQ ID NO: 1. In some embodiments,said at least one non-naturally encoded amino acid is at a positioncorresponding to amino acid 87 in SEQ ID NO: 1. In some embodiments,said at least one non-naturally encoded amino acid is at a positioncorresponding to amino acid 91 in SEQ ID NO: 1. In some embodiments,said at least one non-naturally encoded amino acid is at a positioncorresponding to amino acid 110 in SEQ ID NO: 1. In some embodiments,said at least one non-naturally encoded amino acid is at a positioncorresponding to amino acid 126 in SEQ ID NO: 1. In some embodiments,said at least one non-naturally encoded amino acid is at a positioncorresponding to amino acid 131 in SEQ ID NO: 1. In some embodiments,said at least one non-naturally encoded amino acid is at a positioncorresponding to amino acid 146 in SEQ ID NO: 1. In some embodiments,said at least one non-naturally encoded amino acid is a phenylalaninederivative. In some embodiments, said at least one non-naturally encodedamino acid may be a para-substituted, ortho-substituted, ormeta-substituted phenylalanine derivative. In some embodiments, said atleast one non-naturally encoded amino acid ispara-acetyl-L-phenylalanine. In some embodiments, said at least onenon-naturally encoded amino acid is para-acetyl-L-phenylalanineincorporated at the position corresponding to amino acid 108 in SEQ IDNO:1.

In some embodiments, the disclosure provides a modified FGF-21polypeptide comprising a polypeptide having an amino acid sequenceselected from SEQ ID NOs: 1-7, except that said amino acid sequence maycomprise: (i) an internal deletion of between 2 and 19 amino acids (suchas between 5 and 19 amino acids), wherein said internal deletion iswithin a region corresponding to amino acids 116 to 134 of SEQ ID NO:1,wherein said internal deletion is replaced by a replacement peptidehaving a length of between 0 and 12 amino acids; (ii) 9 or feweradditional amino acid substitutions, deletions, and/or insertions; (iii)a non-naturally encoded amino acid at the position corresponding toamino acid 108 of SEQ ID NO:1, which may comprise a para-substituted,ortho-substituted, or meta-substituted phenylalanine derivate, such aspara-acetyl-L-phenylalanine.

In some embodiments, said modified FGF-21 polypeptide may comprise asubstitution of glutamic acid for glycine at the position correspondingto amino acid 170 of SEQ ID NO: 1.

In some embodiments, said internal deletion may comprise or consist of aregion corresponding to amino acids 119-130 of SEQ ID NO:1. In someembodiments, said modified FGF-21 polypeptide may be linked to apolymer, water soluble polymer, or poly(ethylene glycol), such as apoly(ethylene glycol) having an average molecular weight of about 30kDa. In some embodiments, said replacement peptide has the sequence G,GG, SG, GSG, GGH, or SGG. In some embodiments, said replacement peptidehas the sequence GSG.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycontain 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1,or fewer additional amino acid substitutions, deletions, and/orinsertions. In some embodiments, the modified FGF-21 polypeptidedescribed herein may comprise a polypeptide having an amino acidsequence that may contain 8 or fewer additional amino acidsubstitutions, deletions, and/or insertions. In some embodiments, themodified FGF-21 polypeptide described herein may comprise a polypeptidehaving an amino acid sequence that may contain 7 or fewer additionalamino acid substitutions, deletions, and/or insertions. In someembodiments, the modified FGF-21 polypeptide described herein maycomprise a polypeptide having an amino acid sequence that may contain 6or fewer additional amino acid substitutions, deletions, and/orinsertions. In some embodiments, the modified FGF-21 polypeptidedescribed herein may comprise a polypeptide having an amino acidsequence that may contain 5 or fewer additional amino acidsubstitutions, deletions, and/or insertions. In some embodiments, themodified FGF-21 polypeptide described herein may comprise a polypeptidehaving an amino acid sequence that may contain 4 or fewer additionalamino acid substitutions, deletions, and/or insertions. In someembodiments, the modified FGF-21 polypeptide described herein maycomprise a polypeptide having an amino acid sequence that may contain 3or fewer additional amino acid substitutions, deletions, and/orinsertions. In some embodiments, the modified FGF-21 polypeptidedescribed herein may comprise a polypeptide having an amino acidsequence that may contain 2 or fewer additional amino acidsubstitutions, deletions, and/or insertions. In some embodiments, themodified FGF-21 polypeptide described herein may comprise a polypeptidehaving an amino acid sequence that may contain 1 additional amino acidsubstitution, deletion, and/or insertion. In some embodiments, themodified FGF-21 polypeptide described herein may comprise a polypeptidehaving an amino acid sequence that may contain a substitution ordeletion of the amino acid corresponding to amino acid G170 of SEQ IDNO: 1. In some embodiments, the modified FGF-21 polypeptide describedherein may comprise a polypeptide having an amino acid sequence that maycontain a substitution of glutamic acid for glycine at the positioncorresponding to amino acid 170 of SEQ ID NO: 1. In some embodiments,the modified FGF-21 polypeptide described herein may comprise apolypeptide having an amino acid sequence that may contain no additionalamino acid substitutions, deletions, and/or insertions.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence containing aninternal deletion of between 4 and 19, between 4 and 18, between 4 and17, between 4 and 16 between 4 and 15, between 4 and 14, between 4 and13, between 4 and 12, between 4 and 11, between 4 and 10, between 4 and9, between 4 and 8, between 4 and 7, or between 4 and 6 amino acids. Insome embodiments, said internal deletion may be between 4 and 14 aminoacids. In some embodiments, said internal deletion may be between 4 and12 amino acids. In some embodiments, said internal deletion may bebetween 4 and 10 amino acids. In some embodiments, said internaldeletion may be between 4 and 8 amino acids. In some embodiments, saidinternal deletion may be between 4 and 6 amino acids.

In some embodiments, said internal deletion may be between 5 and 19,between 5 and 18, between 5 and 17, between 5 and 16, between 5 and 15,between 5 and 14, between 5 and 13, between 5 and 12, between 5 and 11,between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, orbetween 5 and 6 amino acids. In some embodiments, said internal deletionmay be between 5 and 14 amino acids. In some embodiments, said internaldeletion may be between 5 and 12 amino acids.

In some embodiments, said internal deletion may be between 6 and 19,between 6 and 18, between 6 and 17, between 6 and 16, between 6 and 15,between 6 and 14, between 6 and 13, between 6 and 12, between 6 and 11,between 6 and 10, between 6 and 9, between 6 and 8, or between 6 and 7amino acids. In some embodiments, said internal deletion may be between6 and 14 amino acids. In some embodiments, said internal deletion may bebetween 6 and 12 amino acids.

In some embodiments, said internal deletion may be between 7 and 19,between 7 and 18, between 7 and 17, between 7 and 16, between 7 and 15,between 7 and 14, between 7 and 13, between 7 and 12, between 7 and 11,between 7 and 10, between 7 and 9, or between 7 and 8 amino acids. Insome embodiments, said internal deletion may be between 7 and 14 aminoacids. In some embodiments, said internal deletion may be between 7 and12 amino acids.

In some embodiments, said internal deletion may be between 8 and 19amino acids. In some embodiments, said internal deletion may be between8 and 14 amino acids. In some embodiments, said internal deletion may bebetween 8 and 12 amino acids.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycontain an internal deletion of 12 amino acids. In some embodiments, themodified FGF-21 polypeptide described herein may comprise a polypeptidehaving an amino acid sequence that may contain an internal deletion of13 amino acids.

In some embodiments, the internal deletion described herein may comprisethe position corresponding to amino acid 121 of SEQ ID NO:1.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycontain an internal deletion, wherein:

i) said internal deletion is within or consists of a regioncorresponding to amino acid 116 to amino acid 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:1;

ii) said internal deletion is within or consists of a regioncorresponding to amino acid 117 to amino acid 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:1;

iii) said internal deletion is within or consists of a regioncorresponding to amino acid118 and to amino acid 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:1;

iv) said internal deletion is within or consists of a regioncorresponding to amino acid 119 to amino acid 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO: 1;

v) said internal deletion is within or consists of a regioncorresponding to amino acid 120 to amino acid 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:1; or

vi) said internal deletion is within or consists of a regioncorresponding to amino acid 121 to amino acid 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:1.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycontain an internal deletion, wherein:

i) said internal deletion is within or consists of a regioncorresponding to amino acid 122 to amino acid 126, 127, 128, 129, 130,131, 132, 133, or 134 of SEQ ID NO:1;

ii) said internal deletion is within or consists of a regioncorresponding to amino acid 123 to amino acid 127, 128, 129, 130, 131,132, 133, or 134 of SEQ ID NO:1;

iii) said internal deletion is within or consists of a regioncorresponding to amino acid 124 to amino acid 128, 129, 130, 131, 132,133, or 134 of SEQ ID NO:1;

iv) said internal deletion is within or consists of a regioncorresponding to amino acid 125 to amino acid 129, 130, 131, 132, 133,or 134 of SEQ ID NO:1;

v) said internal deletion is within or consists of a regioncorresponding to amino acid 126 to amino acid 130, 131, 132, 133, or 134of SEQ ID NO:1;

vi) said internal deletion is within or consists of a regioncorresponding to amino acid 127 to amino acid 131, 132, 133, or 134 ofSEQ ID NO:1;

vii) said internal deletion is within or consists of a regioncorresponding to amino acid 128 to amino acid 132, 133, or 134 of SEQ IDNO:1;

viii) said internal deletion is within or consists of a regioncorresponding to amino acid 129 to amino acid 133, or 134 of SEQ IDNO:1; or

ix) said internal deletion is within or consists of a regioncorresponding to amino acid 130 to amino acid 134 of SEQ ID NO:1.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycontain an internal deletion, wherein:

i) said internal deletion is within or consists of a regioncorresponding to amino acid 116 to amino acid 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 ofSEQ ID NO:1;

ii) said internal deletion is within or consists of a regioncorresponding to amino acid 117 to amino acid 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ IDNO:1;

iii) said internal deletion is within or consists of a regioncorresponding to amino acid 118 to amio acid 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:1;

iv) said internal deletion is within or consists of a regioncorresponding to amino acid 119 to amino acid 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:1;

v) said internal deletion is within or consists of a regioncorresponding to amino acid 120 to amino acid 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:1;

vi) said internal deletion is within or consists of a regioncorresponding to amino acid 121 to amino acid 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:1;

vii) said internal deletion is within or consists of a regioncorresponding to amino acid 122 to amino acid 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, or 134 of SEQ ID NO:1;

viii) said internal deletion is within or consists of a regioncorresponding to amino acid 123 to amino acid 124, 125, 126, 127, 128,129, 130, 131, 132, 133, or 134 of SEQ ID NO:1;

ix) said internal deletion is within or consists of a regioncorresponding to amino acid 124 to amino acid 125, 126, 127, 128, 129,130, 131, 132, 133, or 134 of SEQ ID NO:1;

x) said internal deletion is within or consists of a regioncorresponding to amino acid 125 to amino acid 126, 127, 128, 129, 130,131, 132, 133, or 134 of SEQ ID NO:1;

xi) said internal deletion is within or consists of a regioncorresponding to amino acid 126 to amino acid 127, 128, 129, 130, 131,132, 133, or 134 of SEQ ID NO:1;

xii) said internal deletion is within or consists of a regioncorresponding to amino acid 127 to amino acid 128, 129, 130, 131, 132,133, or 134 of SEQ ID NO:1;

xiii) said internal deletion is within or consists of a regioncorresponding to amino acid 128 to amino acid 129, 130, 131, 132, 133,or 134 of SEQ ID NO:1;

xiv) said internal deletion is within or consists of a regioncorresponding to amino acid 129 to amino acid 130, 131, 132, 133, or 134of SEQ ID NO:1;

xv) said internal deletion is within or consists of a regioncorresponding to amino acid 130 to amino acid 131, 132, 133, or 134 ofSEQ ID NO:1;

xvi) said internal deletion is within or consists of a regioncorresponding to amino acid 131 to amino acid 132, 133, or 134 of SEQ IDNO:1;

xvii) said internal deletion is within or consists of a regioncorresponding to amino acid 132 to amino acid 133, or 134 of SEQ IDNO:1; or

xviii) said internal deletion is within or consists of a regioncorresponding to amino acid 133 to amino acid 134 of SEQ ID NO:1.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycontain an internal deletion, wherein said internal deletion is within aregion corresponding to amino acids 119-130 of SEQ ID NO:1. In someembodiments, the modified FGF-21 polypeptide described herein maycomprise a polypeptide having an amino acid sequence that may contain aninternal deletion, wherein said internal deletion comprises or consistsof a region corresponding to amino acids 119-130 of SEQ ID NO:1.

In some embodiments, the disclosure provides a modified FGF-21polypeptide comprising a polypeptide having an amino acid sequenceselected from SEQ ID NOs: 1-7, except that said amino acid sequence maycomprise: (i) an internal deletion of the region corresponding to aminoacids 119-130 of SEQ ID NO:1, wherein said internal deletion is replacedby a replacement peptide having a length of between 0 and 12 aminoacids; (ii) 9 or fewer additional amino acid substitutions, deletions,and/or insertions; and (iii) a non-naturally encoded amino acid at theposition corresponding to amino acid 108 of SEQ ID NO:1, which maycomprise a para-substituted, ortho-substituted, or meta-substitutedphenylalanine derivate, such as para-acetyl-L-phenylalanine. Saidmodified FGF-21 polypeptide may comprise a substitution of glutamic acidfor glycine at the position corresponding to amino acid 170 of SEQ IDNO: 1. Said modified FGF-21 polypeptide may be linked to a polymer,water soluble polymer, or poly(ethylene glycol), such as a poly(ethyleneglycol) having an average molecular weight of about 30 kDa. In someembodiments, said replacement peptide has the sequence G, GG, SG, GSG,GGH, or SGG. In some embodiments, said replacement peptide has thesequence GSG.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycomprise a replacement polypeptide. In some embodiments, saidreplacement peptide has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or12 amino acids. In some embodiments, said replacement peptide has alength of 2-8 amino acids. In some embodiments, said replacement peptidehas a length of 2-5 amino acids. In some embodiments, said replacementpeptide has a length of 3 amino acids. In some embodiments, saidreplacement peptide comprises serine, histidine, and/or glycineresidues. In some embodiments, said replacement peptide comprises serineand/or glycine residues. In some embodiments, said replacement peptidehas the sequence G, GG, SG, GSG, GGH, or SGG. In some embodiments, saidreplacement peptide has the sequence GSG.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycomprise an internal deletion within the region corresponding to aminoacids 119-130 of SEQ ID NO:1 and a replacement peptide having thesequence GSG.

In some embodiments, the disclosure provides a modified FGF-21polypeptide comprising a polypeptide having an amino acid sequenceselected from SEQ ID NOs: 1-7, except that said amino acid sequence maycomprise: (i) an internal deletion of between 2 and 19 amino acids (suchas between 5 and 19 amino acids), wherein said internal deletion iswithin a region corresponding to amino acids 116 to 134 of SEQ ID NO:1,wherein said internal deletion is replaced by a replacement peptidehaving the sequence G, GG, SG, GSG, GGH, or SGG; (ii) 9 or feweradditional amino acid substitutions, deletions, and/or insertions; (iii)a non-naturally encoded amino acid at the position corresponding toamino acid 108 of SEQ ID NO:1, which may comprise a para-substituted,ortho-substituted, or meta-substituted phenylalanine derivate, such aspara-acetyl-L-phenylalanine. Said modified FGF-21 polypeptide maycomprise a substitution of glutamic acid for glycine at the positioncorresponding to amino acid 170 of SEQ ID NO: 1. Said internal deletionmay comprise or consist of a region corresponding to amino acids 119-130of SEQ ID NO:1. Said modified FGF-21 polypeptide may be linked to apolymer, water soluble polymer, or poly(ethylene glycol), such as apoly(ethylene glycol) having an average molecular weight of about 30kDa. In some embodiments, said replacement peptide has the sequence GSG.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycontain an internal deletion that comprises the region corresponding toamino acids 119-130 of SEQ ID NO:1, a replacement peptide having thesequence GSG, a substitution of glutamic acid for glycine at theposition corresponding to amino acid 170 of SEQ ID NO: 1, and optionallya non-naturally encoded amino acid at the position corresponding toamino acid 108 of SEQ ID NO:1, which may comprise a para-substituted,ortho-substituted, or meta-substituted phenylalanine derivate, such aspara-acetyl-L-phenylalanine.

In some embodiments, the modified FGF-21 polypeptide of described hereinmay comprise a polypeptide having an amino acid sequence that may haveat least 90%, 95%, 96%, 97%, 98%, 99% identity to the polypeptide of SEQID NO: 202 with or without the N-terminal methionine. In someembodiments, said amino acid sequence may have at least 95% identity tothe polypeptide of SEQ ID NO: 202 with or without the N-terminalmethionine. In some embodiments, said amino acid sequence may have atleast 97% identity to the polypeptide of SEQ ID NO: 202 with or withoutthe N-terminal methionine. In some embodiments, said amino acid sequencemay have at least 98% identity to the polypeptide of SEQ ID NO: 202 withor without the N-terminal methionine. In some embodiments, said aminoacid sequence may have at least 99% identity to the polypeptide of SEQID NO: 202 with or without the N-terminal methionine. In someembodiments, said amino acid sequence may comprise or consist of thepolypeptide of SEQ ID NO: 202 with or without the N-terminal methionine.In some embodiments, said amino acid sequence may comprise or consist ofthe polypeptide of SEQ ID NO: 202. In some embodiments, said amino acidsequence may comprise or consist of the polypeptide of SEQ ID NO: 202without the N-terminal methionine.

In some embodiments, the modified FGF-21 polypeptide of described hereinmay comprise a polypeptide having an amino acid sequence that may haveat least 90%, 95%, 96%, 97%, 98%, 99% identity to the polypeptide of SEQID NO: 102 with or without the N-terminal methionine. In someembodiments, said amino acid sequence may have at least 95% identity tothe polypeptide of SEQ ID NO: 102 with or without the N-terminalmethionine. In some embodiments, said amino acid sequence may have atleast 97% identity to the polypeptide of SEQ ID NO: 102 with or withoutthe N-terminal methionine. In some embodiments, said amino acid sequencemay have at least 98% identity to the polypeptide of SEQ ID NO: 102 withor without the N-terminal methionine. In some embodiments, said aminoacid sequence may have at least 99% identity to the polypeptide of SEQID NO: 102 with or without the N-terminal methionine. In someembodiments, said amino acid sequence may comprise or consist of thepolypeptide of SEQ ID NO: 102 with or without the N-terminal methionine.In some embodiments, said amino acid sequence may comprise or consist ofthe polypeptide of SEQ ID NO: 102. In some embodiments, said amino acidsequence may comprise or consist of the polypeptide of SEQ ID NO: 102without the N-terminal methionine.

In some embodiments, the modified FGF-21 polypeptide described hereinmay further comprise a fusion partner. In some embodiments, the modifiedFGF-21 polypeptide described herein may comprise a connecting peptidehaving a length of 0-50 amino acids between said amino acid sequence anda fusion partner. In some embodiments, the connecting peptide may havean amino acid sequence selected from SEQ ID NO:74-100, 301, and 350-383.In some embodiments, the connecting peptide may have the amino acidsequence GGGGGSGGGSGGGGS (SEQ ID NO:360).

In some embodiments, the modified FGF-21 polypeptide may furthercomprise a connecting peptide that links said amino acid sequence andsaid fusion partner. In some embodiments, the connecting peptide mayhave a length of between 0 and 100, between 2 and 80, between 2 and 60,between 2 and 50, between 2 and 40, between 2 and 30, between 2 and 20,between 2 and 10, between 2 and 8, between 2 and 6, or between 2 and 4amino acids. In some embodiments, the connecting peptide may comprise orconsist of an amino acid sequence selected from: SEQ ID NOs:74-100, 301,and 350-383.

In some embodiments, the connecting peptide may comprise or consist ofthe amino acid sequence GGGGGSGGGSGGGGS (SEQ ID NO:360).

In some embodiments, the fusion partner is selected from serum albumin,Fc domain, immunoglobulin constant region, unstructured polypeptide, andadnectin and fragments thereof. In some embodiments, the fusion partnercomprises an immunoglobulin constant region or a modified immunoglobulinconstant region. In some embodiments, the fusion partner may comprise anunstructured polypeptide, wherein said unstructured polypeptidecomprises an XTEN or PAS polypeptide. In some embodiments, the PASpolypeptide may have an amino acid sequence selected from SEQ ID NOs:310-316 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, or95% identical thereto.

In some embodiments, the fusion partner comprises an Fc domain or afragment thereof. In some embodiments, the Fc domain may have an aminoacid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence selected from SEQ ID NOs:302 and323-335, or a fragment thereof, such as a polypeptide selected from SEQID NOs:303-309. In some embodiments, the fusion partner may comprise anFc domain or Fc fragment having an amino acid sequence selected from SEQID NOs: 302 and 323-335. In some embodiments, the Fc domain may have anamino acid sequence at least 95% identical to an amino acid sequenceselected from SEQ ID NOs:302 and 323-335, or a fragment thereof. In someembodiments, the Fc domain may have an amino acid sequence at least 90%identical to SEQ ID NOs:302. In some embodiments, the Fc domain may havean amino acid sequence at least 95% identical to SEQ ID NO:302. In someembodiments, the Fc domain may comprise the amino acid sequence of SEQID NO:302.

In some embodiments, the modified FGF-21 polypeptide may comprises orconsists of a polypeptide at least 90%, 95%, 97%, 98%, 99%, or 100%identical to a polypeptide selected from: SEQ ID NOs:475-487. In someembodiments, the modified FGF-21 polypeptide comprises or consists of apolypeptide at least 90% identical to SEQ ID NO:475. In someembodiments, the modified FGF-21 polypeptide comprises or consists of apolypeptide at least 95% identical to SEQ ID NO:475. In someembodiments, the modified FGF-21 polypeptide comprises or consists of apolypeptide at least 96% identical to SEQ ID NO:475. In someembodiments, the modified FGF-21 polypeptide comprises or consists of apolypeptide at least 97% identical to SEQ ID NO:475. In someembodiments, the modified FGF-21 polypeptide comprises or consists of apolypeptide at least 98% identical to SEQ ID NO:475. In someembodiments, the modified FGF-21 polypeptide comprises or consists of apolypeptide at least 99% identical to SEQ ID NO:475. In someembodiments, the modified FGF-21 polypeptide comprises or consists ofthe polypeptide of SEQ ID NO:475.

In some embodiments, the fusion partner may comprise an adnectin. Insome embodiments, the fusion partner may comprise an albumin-binding orPKE adnectin. In some embodiments, the PKE adnectin may comprise thepolypeptide at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identicalto SEQ ID NO: 320. In some embodiments, the PKE adnectin may comprisethe polypeptide at least 90% identical to SEQ ID NO: 320. In someembodiments, the PKE adnectin may comprise the polypeptide of SEQ IDNO:320. In some embodiments, the modified FGF-21 polypeptide maycomprise or consist of a polypeptide at least 90%, 95%, 97%, 98%, 99%,or 100% identical to a polypeptide selected from: SEQ ID NOs:401-423. Insome embodiments, the modified FGF-21 polypeptide may comprise orconsist of a polypeptide at least 95% identical to a polypeptideselected from: SEQ ID NOs:401-423. In some embodiments, the modifiedFGF-21 polypeptide may comprise or consist of a polypeptide selectedfrom: SEQ ID NOs:401-423.

In some embodiments, the PKE adnectin may comprise the polypeptide atleast 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:319. In some embodiments, the PKE adnectin may comprise the polypeptideat least 90% identical to SEQ ID NO: 319. In some embodiments, the PKEadnectin may comprise the polypeptide of SEQ ID NO:319. In someembodiments, the modified FGF-21 polypeptide may comprise or consist ofa polypeptide at least 90%, 95%, 97%, 98%, 99%, or 100% identical to apolypeptide selected from: SEQ ID NOs: 452-474. In some embodiments, themodified FGF-21 polypeptide may comprise or consist of a polypeptide atleast 95% identical to a polypeptide selected from: SEQ ID NOs: 452-474.In some embodiments, the modified FGF-21 polypeptide may comprise orconsist of a polypeptide selected from: SEQ ID NOs: 452-474.

In some embodiments, the fusion partner comprises serum albumin or afragment thereof. In some embodiments, the fusion partner compriseshuman serum albumin or a fragment thereof. In some embodiments, thefusion partner may comprise a polypeptide at least 90% identical to SEQID NO: 321 or 322. In some embodiments, the fusion partner may comprisethe polypeptide of SEQ ID NO:321 or 322. In some embodiments, themodified FGF-21 polypeptide may comprise a polypeptide at least 90%,95%, 97%, 98%, 99%, or 100% identical to a polypeptide selected from:SEQ ID NOs:424-432, 434-437, 440-443, and 446-451. In some embodiments,the modified FGF-21 polypeptide may comprise a polypeptide at least 95%,identical to a polypeptide selected from: SEQ ID NOs:424-432, 434-437,440-443, and 446-451. In some embodiments, the modified FGF-21polypeptide may comprise a polypeptide selected from: SEQ IDNOs:424-432, 434-437, 440-443, and 446-451.

In some embodiments, the disclosure provides a modified FGF-21polypeptide comprising a polypeptide having an amino acid sequenceselected from SEQ ID NOs: 1-7, except that said amino acid sequence maycomprise: (i) an internal deletion of between 2 and 19 amino acids (suchas between 5 and 19 amino acids), wherein said internal deletion iswithin a region corresponding to amino acids 116 to 134 of SEQ ID NO:1,wherein said internal deletion is replaced by a replacement peptidehaving a length of between 0 and 12 amino acids; and (ii) 9 or feweradditional amino acid substitutions, deletions, and/or insertions;wherein said modified FGF-21 polypeptide further comprises a fusionpartner. Said fusion partner may comprise an Fc domain or a fragmentthereof. Said modified FGF-21 polypeptide may comprise a substitution ofglutamic acid for glycine at the position corresponding to amino acid170 of SEQ ID NO: 1. Said internal deletion may comprise or consist of aregion corresponding to amino acids 119-130 of SEQ ID NO:1. In someembodiments, said replacement peptide has the sequence G, GG, SG, GSG,GGH, or SGG. In some embodiments, said replacement peptide has thesequence GSG. In some embodiments, the modified FGF-21 polypeptidecomprises a connecting peptide between said amino acid sequence and thefusion partner, wherein said connecting peptide comprises or consists ofthe amino acid sequence GGGGGSGGGSGGGGS (SEQ ID NO:360).

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence that maycontain a non-naturally encoded amino acid that may be linked to alinker, polymer, biologically active molecule, peptide, polypeptide, orhalf-life extending moiety. In some embodiments, the modified FGF-21polypeptide described herein may comprise a polypeptide having an aminoacid sequence that may contain a non-naturally encoded amino acid thatmay be linked to a half-life extending moiety. In some embodiments, saidhalf-life extending moiety comprises a water soluble polymer. In someembodiments, said half-life extending moiety is selected frompoly(ethylene glycol) (PEG), monomethoxy PEG (mPEG), an unstructuredpolypeptide, an adnectin, serum albumin, human serum albumin, an Fcdomain, an immunoglobulin constant region, a fragment of any of theforegoing, a lipid, a branched or unbranched acyl group, a branched orunbranched C8-C30 acyl group, a branched or unbranched alkyl group, anda branched or unbranched C8-C30 alkyl group. In some embodiments, themodified FGF-21 polypeptide described herein may comprise a polypeptidehaving an amino acid sequence that may contain a non-naturally encodedamino acid that may be linked to an unstructured polypeptide selectedfrom an XTEN or PAS polypeptide. In some embodiments, said half-lifeextending moiety comprises a poly(ethylene glycol) (PEG) or monomethoxyPEG (mPEG) polymer. In some embodiments, said half-life extending moietycomprises a branched or multiarmed poly(ethylene glycol) (PEG), or otherbranched or multiarmed water soluble polymer. In some embodiments, saidhalf-life extending moiety comprises a poly(ethylene glycol) (PEG) ormonomethoxy PEG (mPEG) moiety having an average molecular weight ofbetween about 0.1 kDa and about 100 kDa. In some embodiments, saidhalf-life extending moiety comprises a poly(ethylene glycol) ormonomethoxy PEG (mPEG) moiety having an average molecular weight:

i) between about 0.1 kDa and about 100 kDa;

ii) between about 1 kDa and 50 kDa;

iii) between about 10 kDa and 40 kDa;

iv) between about 20 kDa and 30 kDa;

v) between about 0.050 kDa and about 100 kDa; or

vi) of about 100 kDa, 95 kDa, 90 kDa, 85 kDa, 80 kDa, 75 kDa, 70 kDa, 65kDa, 60 kDa, 55 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa, 25 kDa, 20kDa, 15 kDa, 10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5 kDa, 4 kDa, 3 kDa, 2kDa, 1 kDa, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, or 100 Da.

In some embodiments, said half-life extending moiety comprises apoly(ethylene glycol) having an average molecular weight of about 30kDa. In some embodiments, said half-life extending moiety comprises anon-poly(ethylene glycol) water soluble polymer or oligosaccharide.

In some embodiments, said non-naturally encoded amino acid describedherein may comprise a carbonyl group, an aminooxy group, a hydrazidegroup, a hydrazine group, a semicarbazide group, an azide group, or analkyne group. In some embodiments, said at least one non-naturallyencoded amino acid comprises a carbonyl moiety and is linked to alinker, polymer, biologically active molecule, or half-life extendingmoiety comprising an aminooxy, a hydrazine, a hydrazide or asemicarbazide moiety. In some embodiments, said at least onenon-naturally encoded amino acid comprises an aminooxy, hydrazine,hydrazide or semicarbazide moiety which is linked to a linker, polymer,biologically active molecule, or half-life extending moiety through anamide linkage. In some embodiments, said at least one non-naturallyencoded amino acid comprises an alkyne moiety which is linked to alinker, polymer, biologically active molecule, or half-life extendingmoiety via an azide moiety. In some embodiments, said at least onenon-naturally encoded amino acid comprises an azide moiety which islinked to a linker, polymer, biologically active molecule, or half-lifeextending moiety comprising an alkyne moiety. In some embodiments, saidat least one non-naturally encoded amino acid comprises an azide oralkyne moiety which is linked to a linker, polymer, biologically activemolecule, or half-life extending moiety through an amide linkage. Insome embodiments, said at least one non-naturally encoded amino acid islinked to a linker, polymer, biologically active molecule, or half-lifeextending moiety through an oxime linkage. In some embodiments, said atleast one non-naturally encoded amino acid is linked to a linker,polymer, biologically active molecule, or half-life extending moietythrough an oxime linkage, wherein said oxime linkage has the structureresulting from the reaction of a carbonyl group and aminooxy group. Insome embodiments, said at least one non-naturally encoded amino acid islinked to a linker, polymer, biologically active molecule, or half-lifeextending moiety through an oxime linkage, wherein said oxime linkagehas the structure resulting from the reaction of a carbonyl groupcontained in said non-naturally encoded amino acid and aminooxy groupcontained in said linker, polymer, biologically active molecule, orhalf-life extending moiety.

In some embodiments, the modified FGF-21 polypeptide described hereinpossesses at least one biological activity of the wild-type human FGF-21polypeptide having the amino acid sequence of SEQ ID NO:1. In someembodiments, the modified FGF-21 polypeptide described herein possessesat least one of increased thermal stability, reduced aggregation,decreased in vivo proteolysis, decreased deamidation, and increasedsolubility compared to a FGF-21 polypeptide without said internaldeletion or a FGF-21 polypeptide that comprises the amino acid sequenceof SEQ ID NO:1 or SEQ ID NO: 201, or compared to the same modifiedFGF-21 polypeptide without said internal deletion and replacementpeptide. In some embodiments, the modified FGF-21 polypeptide describedherein possesses at least one of increased thermal stability, reducedaggregation, decreased in vivo proteolysis, decreased deamidation, andincreased solubility compared to a pegylated FGF-21 polypeptide withoutsaid internal deletion, a non-pegylated FGF-21 polypeptide without saidinternal deletion, a FGF-21 polypeptide that comprises the amino acidsequence of SEQ ID NO:1, a non-pegylated FGF-21 polypeptide thatcomprises the amino acid sequence of SEQ ID NO: 201, pegylated SEQ IDNO: 201, or compared to the same modified FGF-21 polypeptide withoutsaid internal deletion and replacement peptide.

In some embodiments, the modified FGF-21 polypeptide may be compared toa pegylated FGF-21 polypeptide without said internal deletion. In someembodiments, the modified FGF-21 polypeptide may be compared to anon-pegylated FGF-21 polypeptide without said internal deletion. In someembodiments, the modified FGF-21 polypeptide may be compared to a FGF-21polypeptide that comprises the amino acid sequence of SEQ ID NO:1. Insome embodiments, the modified FGF-21 polypeptide may be compared to SEQID NO: 201. In some embodiments, the modified FGF-21 polypeptide may becompared to a pegylated SEQ ID NO: 201. In some embodiments, themodified FGF-21 polypeptide may be compared to the same modified FGF-21polypeptide without said internal deletion and replacement peptide.

In some embodiments, the modified FGF-21 polypeptide described hereinexhibits an increase in transition midpoint (melting temperature, Tm) ofbetween 2° C. and 12° C., between 2° C. and 10° C., between 2° C. and 8°C., between 4° C. and 8° C., between 4° C. and 10° C., between 4° C. and12° C., or between 6° C. and 8° C. compared to an unmodified FGF-21polypeptide that comprises the amino acid sequence of SEQ ID NO:1 or SEQID NO: 201 or a FGF-21 polypeptide without said internal deletion.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence having at least85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the polypeptide ofSEQ ID NO: 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 202, 205, 206, 210, 211, 212, 219, 220, 221,222, or 223. In some embodiments, the modified FGF-21 polypeptidedescribed herein may comprise a polypeptide having an amino acidsequence having at least 95% identity to the polypeptide of SEQ ID NO:102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 202, 205, 206, 210, 211, 212, 219, 220, 221, 222, or 223.In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence having at least98% identity to the polypeptide of SEQ ID NO: 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 202, 205,206, 210, 211, 212, 219, 220, 221, 222, or 223. In some embodiments, themodified FGF-21 polypeptide described herein may comprise a polypeptidehaving an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identity to the polypeptide of SEQ ID NO: 102 or SEQ IDNO: 202. In some embodiments, the modified FGF-21 polypeptide describedherein may comprise a polypeptide having an amino acid sequence havingat least 95% identity to the polypeptide of SEQ ID NO: 102 or SEQ ID NO:202. In some embodiments, the modified FGF-21 polypeptide describedherein may comprise a polypeptide having an amino acid sequence havingat least 98% identity to the polypeptide of SEQ ID NO: 102 or SEQ ID NO:202. In some embodiments, the modified FGF-21 polypeptide describedherein may comprise a polypeptide having an amino acid sequence of SEQID NO: 202. In some embodiments, the modified FGF-21 polypeptidedescribed herein may comprise a polypeptide having an amino acidsequence of SEQ ID NO: 202, wherein pAF in SEQ ID NO:202 may be linkedto a poly(ethylene glycol). In some embodiments, the poly(ethyleneglycol) may have an average molecular weight of about 30 kDa.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an N-terminal methionine (which may bepresent after expression in some systems, such as E. coli). In someembodiments, the modified FGF-21 polypeptide described herein maycomprise a polypeptide having no N-terminal methionine, for example as aresult of processing to remove a signal peptide containing an N-terminalmethionine, e.g. in a mammalian cell-based expression system. In someembodiments, the modified FGF-21 polypeptide may include any of thesequences or variants thereof disclosed herein but omitting anN-terminal methionine, e.g., any one of SEQ ID NOs:101-132, 201-202,205-206, 210-212, 219-223, or any one of the variant, modified, orfusion forms thereof described herein, with the N-terminal methionineomitted or absent. In some embodiments, the modified FGF-21 polypeptidemay include any of the sequences or variants thereof disclosed herein,e.g., any one of SEQ ID NOs:401-487 or any one of the variant, modified,or fusion forms thereof described herein, with an N-terminal methionineadded or present. In some embodiments, the modified FGF-21 polypeptidedescribed herein may comprise a polypeptide having an amino acidsequence of SEQ ID NO: 202 without the N-terminal methionine. In someembodiments, the modified FGF-21 polypeptide described herein maycomprise a polypeptide having an amino acid sequence of SEQ ID NO: 202(optionally without the N-terminal methionine), wherein the pAF in SEQID NO:202 is linked to a poly(ethylene glycol) having an averagemolecular weight of about 30 kDa.

In some embodiments, the modified FGF-21 polypeptide described hereinmay comprise a polypeptide having an amino acid sequence of SEQ ID NO:102. In some embodiments, the modified FGF-21 polypeptide describedherein may comprise a polypeptide having an amino acid sequence of SEQID NO: 102 without the N-terminal methionine. In some embodiments, themodified FGF-21 polypeptide described herein may comprise a polypeptidehaving an amino acid sequence of SEQ ID NO: 102 without the N-terminalmethionine and a Fc domain or fragment thereof. In some embodiments, themodified FGF-21 polypeptide described herein may comprise a polypeptidehaving an amino acid sequence of SEQ ID NO: 102 and a Fc domain orfragment thereof. In some embodiments, the modified FGF-21 polypeptidedescribed herein may comprise SEQ ID NO:475.

In some embodiments the disclosure provides an isolated nucleic acidencoding a modified FGF-21 polypeptide described herein. In someembodiments, provided herein is an expression vector comprising anisolated nucleic acid encoding a modified FGF-21 polypeptide describedherein. In some embodiments, provided herein is a host cell comprisingan expression vector comprising an isolated nucleic acid encoding amodified FGF-21 polypeptide described herein. In some embodiments, thehost cell is a mammalian cell, such as CHO or HEK. In some embodiments,the host cell is a bacterium, such as E. coli. In some embodiments, thehost cell is a yeast, such as Saccharomyces cerevisiae. In someembodiments the disclosure provides a method of producing a modifiedFGF-21 polypeptide, comprising culturing the host cell described hereinand isolating said modified FGF-21 polypeptide. In some embodiments thedisclosure provides a method of producing a modified FGF-21 polypeptidecomprising a non-naturally encoded amino acid. In some embodiments, thenon-naturally encoded amino acid is encoded by a selector codon. In someembodiments, the method comprises culturing a host cell described hereinwherein said host cell comprises an orthogonal tRNA that recognizes saidselector codon and introduces said non-naturally encoded amino acid intosaid modified FGF-21 polypeptide, and isolating said modified FGF-21polypeptide.

In some embodiments the disclosure provides a composition comprising themodified FGF-21 polypeptide described herein and a pharmaceuticallyacceptable carrier or excipient. In some embodiments the disclosureprovides a composition comprising a modified FGF-21 polypeptidecomprising a polypeptide having an amino acid sequence of SEQ ID NO:202, wherein pAF in SEQ ID NO:202 may be linked to a poly(ethyleneglycol), and a pharmaceutically acceptable carrier or excipient. In someembodiments the poly(ethylene glycol) has an average molecular weight ofabout 30 kDa.

In some embodiments, the disclosure provides a composition comprising amodified FGF-21 polypeptide comprising a polypeptide having an aminoacid sequence of SEQ ID NO: 102 without the N-terminal methionine, fusedto a Fc domain or fragment thereof, and a pharmaceutically acceptablecarrier or excipient. In some embodiments, the disclosure provides acomposition comprising a modified FGF-21 polypeptide comprising SEQ IDNO:475 and a pharmaceutically acceptable carrier or excipient.

In some embodiments the disclosure provides a composition comprising themodified FGF-21 polypeptide described herein and a pharmaceuticallyacceptable carrier or excipient and at least one other active agent. Insome embodiments, the at least one other active agent is ananti-diabetes agent, cholesterol controlling agent, anti-inflammatoryagent, anti-obesity agent, an antihypertensive agent, or ananti-fibrosis agent. In some embodiments, the at least one other activeagent is a GLP-1 agonist or an insulin. In some embodiments, the atleast one other active agent is a rapid acting, short acting, regularacting, intermediate acting, or long acting insulin, Humalog, Lispro,Novolog, Apidra, Humulin, Aspart, human insulin, NPH, Lente, Ultralente,Lantus, Glargine, Levemir, Detemirm; exenatide (Byetta/Bydureon),liraglutide (Victoza), lixisenatide (Lyxumia), albiglutide (Tanzeum),exenatide long-acting release (LAR), taspoglutide, albiglutide,LY2189265 (Dulaglutide); orlistat (Xenical), a pancreatic lipaseinhibitor, naltrexone, phentermine, topiramate (Qsymia), lorcaserin(Belviq), naltrexone and bupropion (Contravene), rimonabant (Acomplia),a cannabinoid receptor antagonist, sibutramine (Meridia), lorcaserin,rimonabant, pramlintide, phentermine, topiramate, bupropion, orglucomannan.

In some embodiments the disclosure provides a method of regulating atleast one of glucose and lipid homeostasis, glucose uptake, GLUT 1expression, and/or serum concentrations of glucose, triglycerides,insulin or glucagon in a patient in need thereof, comprisingadministering to the patient a therapeutically effective amount of amodified FGF-21 polypeptide disclosed herein or a composition disclosedherein. In some embodiments the disclosure provides a method ofincreasing insulin sensitivity, increasing levels of adiponectin,reducing levels of blood glucose, reducing levels of glucagon, reducinglevels of triglyceride, reducing levels of fructosamine, reducing levelsof low density cholesterol, or reducing levels of C-reactive protein ina patient in need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedisclosed herein or a composition disclosed herein. In some embodimentsthe disclosure provides a method of treating a condition or disorderselected from obesity, diabetes, pancreatitis, insulin resistance,hyperinsulinemia, glucose intolerance, hyperglycemia, metabolicsyndrome, impaired glucose tolerance, inadequate glucose clearance, highblood glucose, and Prader-Willi syndrome in a patient in need thereof,comprising administering to the patient a therapeutically effectiveamount of a modified FGF-21 polypeptide disclosed herein or acomposition disclosed herein. In some embodiments the disclosureprovides a method of treating an insulin related condition or disorderselected from Type A Insulin Resistance, Type C Insulin Resistance (AKAHAIR-AN Syndrome), Rabson-Mendenhall Syndrome, Donohue's Syndrome orLeprechaunism, hyperandrogenism, hirsuitism, or acanthosis nigricans ina patient in need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedisclosed herein or a composition disclosed herein. In some embodimentsthe disclosure provides a method of treating type 1 diabetes or type 2diabetes in a patient in need thereof, comprising administering to thepatient a therapeutically effective amount of a modified FGF-21polypeptide disclosed herein or a composition disclosed herein. In someembodiments the disclosure provides a method of treating obesity in apatient in need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidedisclosed herein or a composition disclosed herein.

In another embodiment, the present disclosure provides a method oftreating a disease associated with fibrosis comprising administering toa patient in need thereof an effective amount of a modified FGF-21polypeptide or a composition comprising a modified FGF-21 polypeptide asdescribed herein. In some embodiments, the disease associated withfibrosis may affect an organ or tissue such as the pancreas, lung,heart, kidney, liver, eyes, nervous system, bone marrow, lymph nodes,endomyocardium, and/or retroperitoneum. In some embodiments, the diseaseassociated with fibrosis may be liver fibrosis or pre-cirrhosis. In someembodiments, the disease associated with fibrosis may be selected from:nonalcoholic steatohepatitis (NASH), cirrhosis, diffuse parenchymal lungdisease, cystic fibrosis, pulmonary fibrosis, progressive massivefibrosis, idiopathic pulmonary fibrosis, injection fibrosis, renalfibrosis, chronic kidney disease, diabetic kidney disease, focalsegmental glomerulosclerosis, membranous nephropathy, IgA nephropathy,myelofibrosis, heart failure, metabolic heart failure, cardiac fibrosis,cataract fibrosis, cataract, ocular scarring, pancreatic fibrosis, skinfibrosis, intestinal fibrosis, intestinal strictures, endomyocardialfibrosis, atrial fibrosis, mediastinal fibrosis, Crohn's disease,retroperitoneal fibrosis, keloid, nephrogenic systemic fibrosis,scleroderma, systemic sclerosis, arthrofibrosis, Peyronie's syndrome,Dupuytren's contracture, diabetic neuropathy, adhesive capsulitis,alcoholic liver disease, hepatosteatosis, viral hepatitis, biliarydisease, primary hemochromatosis, drug-related cirrhosis, cryptogeniccirrhosis, Wilson's disease, and, alpha 1-antitrypsin deficiency,interstitial lung disease (ILD), human fibrotic lung disease, liverfibrosis, macular degeneration, retinal retinopathy, vitrealretinopathy, myocardial fibrosis, Grave's ophthalmopathy, drug inducedergotism, cardiovascular disease, atherosclerosis/restenosis,hypertrophic scars, primary or idiopathic myelofibrosis, andinflammatory bowel disease (including, but not limited to, collagenouscolitis). In some embodiments, the disease associated with fibrosisresults from one or more of pulmonary disease, lung cancer, drugtherapy, chemotherapy, or radiation therapy. In some embodiments, thedisease associated with fibrosis results from one or more of aging,heart attack, stroke, myocardial damage, or left ventriculardysfunction. In some embodiments, the disease associated with fibrosismay be selected from renal fibrosis, glomerular nephritis, chronickidney disease, chronic kidney failure, and nephritis associated withsystemic lupus, cancer, physical obstructions, toxins, metabolicdisease, immunological diseases, or diabetic nephropathy. In someembodiments, the disease associated with fibrosis results from one ormore of trauma, spinal injury, infection, surgery, ischemic injury,heart attack, burns, environmental pollutant exposure, pneumonia,tuberculosis, or acute respiratory distress syndrome. In someembodiments, the disease associated with fibrosis may be selected frompulmonary fibrosis, interstitial lung disease, human fibrotic lungdisease, idiopathic pulmonary fibrosis, liver fibrosis, cardiacfibrosis, myocardial fibrosis, macular degeneration, retinalretinopathy, vitreal retinopathy, Grave's ophthalmopathy, drug inducedergotism, cardiovascular disease, atherosclerosis/restenosis, keloidsand hypertrophic scars, primary or idiopathic myelofibrosis,inflammatory bowel disease, collagenous colitis, ocular scarring andcataract fibrosis. In some embodiments, the disease associated withfibrosis may be selected from NASH, liver fibrosis, and cirrhosis. Insome embodiments, the disease associated with fibrosis may be NASH. Insome embodiments, the disease associated with fibrosis may be selectedfrom diabetic kidney disease, chronic kidney disease, and renalfibrosis. In some embodiments, the disease associated with fibrosis maybe selected from metabolic heart failure and cardiac fibrosis. In someembodiments, the disease associated with fibrosis may be lung fibrosis.

In some embodiments, the present disclosure provides a method oftreating liver fibrosis or cirrhosis in a patient in need thereof,comprising administering to the patient an effective amount of amodified FGF-21 polypeptide described herein or a composition describedherein. In some embodiments, the present disclosure provides a method oftreating or preventing NASH in a patient in need thereof, comprisingadministering to the patient an effective amount of a modified FGF-21polypeptide described herein or a composition described herein.

In some embodiments, the present disclosure provides a method ofdecreasing the hepatic fat fraction in a patient in need thereof,comprising administering to the patient an effective amount of amodified FGF-21 polypeptide described herein or a composition describedherein, wherein optionally said patient is at risk of developing or hasbeen diagnosed with NASH. In some embodiments, the present disclosureprovides a method of decreasing liver stiffness, decreasing percentagebody fat, decreasing body weight, decreasing liver-to-body weight ratio,decreasing liver lipid content, decreasing liver fibrosis area,decreasing fasting blood glucose levels, fasting triglyceride,decreasing LDL cholesterol, decreasing ApoB, decreasing ApoC, and/orincreasing HDL cholesterol in a patient in need thereof, comprisingadministering to the patient an effective amount of a modified FGF-21polypeptide described herein or a composition described herein, whereinoptionally said patient is at risk of developing or has been diagnosedwith NASH. In some embodiments, the present disclosure provides a methodof increasing adiponectin levels in a patient in need thereof,comprising administering to the patient an effective amount of amodified FGF-21 polypeptide described herein or a composition describedherein, wherein optionally said patient is at risk of developing or hasbeen diagnosed with NASH. In some embodiments, the present disclosureprovides a method of treating one or more symptoms associated with NASHin a patient in need thereof, comprising administering to the patient aneffective amount of a modified FGF-21 polypeptide described herein or acomposition described herein.

Provided herein are methods of treating or preventing NASH in a patientin need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidecomprising a polypeptide having an amino acid sequence at least 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 202. Insome embodiments, the modified FGF-21 polypeptide comprises apolypeptide having an amino acid sequence at least 95% identical to SEQID NO: 202. Provided herein are also methods of treating or preventingNASH in a patient in need thereof, comprising administering to thepatient a therapeutically effective amount of a modified FGF-21polypeptide comprising SEQ ID NO: 202, wherein the pAF residue thereofis linked to a poly(ethylene glycol) moiety. In some embodiments, saidpoly(ethylene glycol) has a molecular weight of between about 0.1 kDaand 100 kDa, or between about 20 kDa and about 40 kDa, or of about 30kDa. In some embodiments, said poly(ethylene glycol) has a molecularweight of about 30 kDa.

Provided herein are methods of treating or preventing NASH in a patientin need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidecomprising a polypeptide having an amino acid sequence at least 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:102. In someembodiments, the modified FGF-21 polypeptide comprises a polypeptidehaving an amino acid sequence at least 95% identical to SEQ ID NO: 102.Provided herein are also methods of treating or preventing NASH in apatient in need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidecomprising (a) SEQ ID NO:102 without the N-terminal Met, or (b) SEQ IDNO:102.

Provided herein are methods of treating or preventing NASH in a patientin need thereof, comprising administering to the patient atherapeutically effective amount of a modified FGF-21 polypeptidecomprising a polypeptide having an amino acid sequence at least 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 475. Insome embodiments, the modified FGF-21 polypeptide comprises apolypeptide having an amino acid sequence at least 95% identical to SEQID NO: 475. Provided herein are methods of treating or preventing NASHin a patient in need thereof, comprising administering to a patient inneed thereof a therapeutically effective amount of a modified FGF-21polypeptide comprising SEQ ID NO: 475.

In some embodiments, the patient may exhibit NASH CRN fibrosis stage1-3, which optionally is determined by a liver biopsy. In someembodiments, prior to treatment the patient may exhibit a fatty liverindex of at least about 60. In some embodiments, prior to treatment thepatient may exhibit a hepatic fat fraction percentage of at least 10%,which optionally is determined by magnetic resonance imaging.

In some embodiments, the present disclosure provides a method oftreating heart failure or cardiac fibrosis in a patient in need thereof,comprising administering to the patient an effective amount of amodified FGF-21 polypeptide described herein or a composition describedherein. In some embodiments, the present disclosure provides a method oftreating kidney or renal fibrosis in a patient in need thereof,comprising administering to the patient an effective amount of amodified FGF-21 polypeptide described herein or a composition describedherein. In some embodiments, the present disclosure provides a method oftreating lung fibrosis in a patient in need thereof, comprisingadministering to the patient an effective amount of a modified FGF-21polypeptide described herein or a composition described herein.

In some embodiments the present disclosure provides methods of treatinga disease associated with fibrosis in a patient in need thereof,comprising administering to the patient an effective amount of amodified FGF-21 polypeptide comprising one or more non-naturally encodedamino acids, wherein said modified FGF-21 polypeptide possesses at least90% or 95% identity to a human FGF-21 polypeptide having an amino acidsequence selected from SEQ ID NOs:1-7 and 201, wherein said diseaseassociated with fibrosis is selected from NASH, liver fibrosis, diabetickidney disease, chronic kidney disease, renal fibrosis, lung fibrosis,cardiac fibrosis, heart failure, and metabolic heart failure.

In some embodiments, the modified FGF-21 polypeptide possesses at least96%, 97%, 98% or 99% identity to a human FGF-21 polypeptide having anamino acid sequence selected from SEQ ID NOs:1-7 and 201.

In some embodiments the present disclosure provides a method of treatinga disease associated with fibrosis comprising administering to a patientin need thereof an effective amount of the modified FGF-21 polypeptideof SEQ ID NO:201, optionally linked to a polymer or water solublepolymer which may comprise poly(ethylene glycol), optionally having amolecular weight of between 1 and 100 kDa or about 30 kDa, wherein saiddisease associated with fibrosis may be selected from NASH, liverfibrosis, diabetic kidney disease, chronic kidney disease and metabolicheart failure.

In some embodiments, the at least one non-naturally encoded amino acidmay be at a position corresponding to amino acid 72, 77, 86, 87, 91,108, 110, 126, 131, or 146 of SEQ ID NO: 1. In some embodiments, the atleast one non-naturally encoded amino acid may be at a positioncorresponding to amino acid 108 in SEQ ID NO: 1. In some embodiments,the at least one non-naturally encoded amino acid may be at a positioncorresponding to amino acid 77, 91, or 131 in SEQ ID NO:1.

In some embodiments, the non-naturally encoded amino acid may comprise aphenylalanine analog or derivative. In some embodiments, thenon-naturally encoded amino acid may comprisepara-acetyl-L-phenylalanine. In some embodiments, the non-naturallyencoded amino acid may comprise para-acetyl-L-phenylalanine and may beat a position corresponding to amino acid 108 in SEQ ID NO: 1.

In some embodiments, the at least one non-naturally encoded amino acidmay be linked to a poly(ethylene glycol) (PEG) or monomethoxy PEG (mPEG)moiety having an average molecular weight of between about 0.1 kDa andabout 100 kDa. In some embodiments, the at least one non-naturallyencoded amino acid may be linked to a poly(ethylene glycol) ormonomethoxy PEG (mPEG) moiety having an average molecular weight: i)between about 0.1 kDa and about 100 kDa; ii) between about 1 kDa and 50kDa; iii) between about 10 kDa and 40 kDa; iv) between about 20 kDa and30 kDa; v) between about 0.050 kDa and about 100 kDa; or vi) of about100 kDa, 95 kDa, 90 kDa, 85 kDa, 80 kDa, 75 kDa, 70 kDa, 65 kDa, 60 kDa,55 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa, 25 kDa, 20 kDa, 15 kDa,10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5 kDa, 4 kDa, 3 kDa, 2 kDa, 1 kDa,900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100Da. In some embodiments, the at least one non-naturally encoded aminoacid may be linked to a poly(ethylene glycol) having an averagemolecular weight of about 30 kDa.

In some embodiments, the at least one non-naturally encoded amino acidmay be linked to a linker, polymer, biologically active molecule, orhalf-life extending moiety through an oxime linkage. In someembodiments, the oxime linkage has the structure resulting from thereaction of a carbonyl group and aminooxy group.

In some embodiments, the modified FGF 21 polypeptide may possess atleast one biological activity of the wild-type human FGF 21 polypeptidehaving the amino acid sequence of SEQ ID NO:1 or of another FGF-21polypeptide.

In some embodiments, the method may further comprise administration ofat least one other active agent to said patient, wherein said additionalactive agent may be contained in the same composition as said modifiedFGF-21 polypeptide or may be administrated separately. In someembodiments, the at least one other active agent may be selected fromanti-fibrotic agents, N-cadherin antagonist, anti-N cadherin antibody,small molecule N-cadherin antagonist, antagonistic N-cadherin fragment,anti-inflammatory agents, hepatoprotective agents suppressingrenin-angiotensin system (RAS) system, probiotics, and polyunsaturatedfatty acids (PUFAs). In some embodiments, the anti-fibrotic agent may beselected from nintedanib, Pirfenidone, LPA1 antagonists, LPA1 receptorantagonists, GLP1 analog, tralokinumab (IL-13, AstraZeneca), vismodegib(hedgehog antagonist, Roche), PRM-151 (pentraxin-2, TGF beta-1,Promedior), SAR-156597 (bispecific Mab IL-4&IL-13, Sanofi), simtuzumab(anti-lysyl oxidase-like 2 (anti-LOXL2) antibody, Gilead), CKD-942,PTL-202 (PDE inh./pentoxifylline/NAC oral control. release, PacificTher.), omipalisib (oral PI3K/mTOR inhibitor, GSK), IW-001 (oral sol.bovine type V collagen mod., ImmuneWorks), STX-100 (integrin alphaV/beta-6 ant, Stromedix/Biogen), Actimmune (IFN gamma), PC-SOD(midismase; inhaled, LTT Bio-Pharma/CKD Pharm), lebrikizumab (anti-IL-13SC humanized mAb, Roche), AQX-1125 (SHIP1 activator, Aquinox), CC-539(JNK inhibitor, Celgene), FG-3019 (FibroGen), and SAR-100842 (Sanofi).In some embodiments, the hepatoprotective agent may be ursodeoxycholicacid (UDCA) or obeticholic acid (OCA or INT-747, Intercept).

In some embodiments, the present disclosure provides a compositioncomprising a modified FGF-21 polypeptide adapted for use in the methodof treating a disease associated with fibrosis as described herein and apharmaceutically acceptable carrier or excipient. In some embodiments,the composition may further comprise at least one other active agent. Insome embodiments, the at least one other active agent may be selectedfrom anti-fibrotic agents, Pirfenidone, N-cadherin antagonist, anti-Ncadherin antibody, small molecule N-cadherin antagonist, antagonisticN-cadherin fragment, anti-inflammatory agents, hepatoprotective agentssuch as ursodeoxycholic acid (UDCA), obeticholic acid (OCA or INT-747,Intercept), suppressing renin-angiotensin system (RAS) system,probiotics and polyunsaturated fatty acids (PUFAs).

In some embodiments the present disclosure provides a method of treatingmedically complicated obesity comprising administering to a patient inneed thereof an effective amount of a modified FGF-21 polypeptideaccording as described herein or a composition comprising a modifiedFGF-21 polypeptide as described herein. In some embodiments themedically complicated obesity may be associated with Prader-WilliSyndrome.

In some embodiments, the modified FGF-21 polypeptide disclosed herein orcomposition comprising a modified FGF-21 polypeptide disclosed hereinand a pharmaceutically acceptable carrier or excipient may beadministered orally, topically, or via injection. In some embodiments,the modified FGF-21 polypeptide or composition may be administered viasubcutaneous injection, IV injection, intraperitoneal injection,intramuscular injection.

In some embodiments, the modified FGF-21 polypeptide or compositiondisclosed herein is administered at a frequency of about once per day,or less frequently than about once per day. In some embodiments, themodified FGF-21 polypeptide or composition disclosed herein isadministered at a frequency of about twice per week, or less frequentlythan about twice per week. In some embodiments, the modified FGF-21polypeptide or composition disclosed herein is administered at afrequency of about once per week, or less frequently than about twiceper week. In some embodiments, the modified FGF-21 polypeptide orcomposition disclosed herein is administered at a frequency of aboutonce per two weeks, or less frequently than about twice per week. Insome embodiments, the modified FGF-21 polypeptide or compositiondisclosed herein is administered at a frequency of about once per threeweeks, or less frequently than about twice per week. In someembodiments, the modified FGF-21 polypeptide or composition disclosedherein is administered at a frequency of about once per month, or lessfrequently than about once per month. In some embodiments, the modifiedFGF-21 polypeptide or composition disclosed herein is administered at afrequency of once per four weeks. In some embodiments, the modifiedFGF-21 polypeptide or composition disclosed herein is administered at afrequency of about once per day. In some embodiments, the modifiedFGF-21 polypeptide or composition disclosed herein is administered at afrequency of about once per week.

In some embodiments, the modified FGF-21 polypeptide disclosed hereinmay be administered in an amount between about 0.01 mg and about 500 mgper dose, between about 0.1 mg and about 200 mg per dose, between about0.2 mg and about 100 mg per dose, between about 0.5 mg and about 80 mgper dose, between about 1 mg and about 60 mg per dose, between about 5mg and about 40 mg per dose, between about 10 mg and about 30 mg perdose, between about 10 mg and about 20 mg per dose, between about 0.2 mgand about 1 mg per dose, between about 1 mg and about 2 mg per dose,between about 2 mg and about 4 mg per dose, between about 4 mg and about6 mg per dose, between about 6 mg and about 10 mg per dose, betweenabout 10 mg and about 20 mg per dose, between about 20 mg and about 40mg per dose, between about 40 mg and about 60 mg per dose, between about60 mg and about 80 mg per dose, between about 80 mg and about 100 mg perdose, between about 100 mg and about 120 mg per dose, between about 120mg and about 140 mg per dose, between about 140 mg and about 160 mg perdose, between about 160 mg and about 180 mg per dose, between about 180mg and about 200 mg per dose, or between about 200 mg and about 240 mgper dose.

In some embodiments, the modified FGF-21 polypeptide disclosed hereinmay be administered in an amount selected from about 0.2 mg, about 0.6mg, about 1 mg, about 2 mg, about 4 mg, about 6 mg, about 8 mg, about 10mg, about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg,about 120 mg, about 140 mg, about 160 mg, about 180 mg, and about 200 mgper dose. In some embodiments, the modified FGF-21 polypeptide disclosedherein may be administered in an amount selected from about 0.2 mg,about 0.6 mg, about 2 mg, about 6 mg, about 20 mg, about 40 mg, andabout 60 mg per dose.

In some embodiments, the modified FGF-21 polypeptide or compositioncomprising a modified FGF-21 polypeptide disclosed herein and apharmaceutically acceptable carrier or excipient may be co-administered,or administered separately (concurrently or sequentially) with at leastone other active agent. In some embodiments, the at least one otheractive agent is selected from anti-diabetes agents, anti-obesity agents,cholesterol controlling agents, anti-inflammatory agents, andantihypertensive agents.

In some embodiments, the at least one other active agent is selectedfrom a statin, a GLP-1 agonist, and insulin. In some embodiments, the atleast one other active agent is selected from amylin, amylin analog,alpha-glucosidase inhibitor such as miglitol, acarbose, voglibose,metformin, biguanide, a glitazone such as rosiglitazone, pioglitazone,troglitazone, a secretagogue such as exenatide, liraglutide,taspoglutide or lixisenatide, a glycosuric, a dipeptidyl peptidase-4inhibitor, insulin, a rapid acting, short acting, regular acting,intermediate acting, or long acting insulin, Humalog, Lispro, Novolog,Apidra, Humulin, Aspart, human insulin, NPH, Lente, Ultralente, Lantus,Glargine, Levemir, Detemir, Atorvastatin, Cerivastatin, Fluvastatin,Lovastatin, Mevastatin, Pitavastatin, Pravastatin, Rosuvastatin,Simvastatin Vytorin, Advicor, Besylate Caduet, Simcor, orlistat(Xenical), a pancreatic lipase inhibitor, naltrexone, phentermine andtopiramate (Qsymia), lorcaserin (Belviq), naltrexone, bupropion(Contravene), rimonabant (Acomplia), a cannabinoid receptor antagonist,sibutramine (Meridia), lorcaserin, rimonabant, exenatide, pramlintide,phentermine, topiramate, a mood stabilizer, bupropion, glucomannan, guargum, Dexedrine, digoxin, an anorectic drug, an anti-obesity drug,exenatide (Byetta/Bydureon), liraglutide (Victoza), lixisenatide(Lyxumia), albiglutide (Tanzeum), exenatide long-acting release (LAR),taspoglutide, albiglutide, and LY2189265 (Dulaglutide).

Advantageously, modified FGF-21 compounds described herein may increaseefficacy by allowing for a longer circulating half-life requiring fewerdoses, increasing both the convenience to a subject in need of suchtherapy and the likelihood of a subject's compliance with dosingrequirements.

Metabolic syndrome is typically diagnosed in patients exhibiting atleast three of the following signs: abdominal fat—in most men, a 40-inchwaist or greater; high blood sugar—at least 110 milligrams per deciliter(mg/dL) after fasting; high triglycerides—at least 150 mg/dL in thebloodstream; low HDL—less than 40 mg/dL; and, blood pressure of 130/85or higher.

The subject methods may be effective to treat or delay the onset of typeII diabetes and/or obesity in a patient in need thereof. Said patientmay have been diagnosed with pre-diabetes or may exhibit one or morerisk factors for development of type II diabetes, such as a familyhistory of type II diabetes; one or more parents or siblings previouslydiagnosed with type II diabetes; dyslipidemia; total blood triglyceridelevels of at least 200 mg/dL; blood high density lipoprotein level lessthan 35 mg/dL; obesity; body mass index greater than 25 kg/m2; historyof gestational diabetes; previously gave birth to an infant with birthweight greater than 9 lbs.; hypertension; systolic blood pressure of atleast 140 mmHg; diastolic blood pressure of at least 90 mmHg; previousmeasurement of fasting blood glucose of at least 99 mg/dL; vasculardisease; Polycystic Ovarian Syndrome; or acanthosis nigricans.

The patient may exhibit one or more symptoms of pre-diabetes such asfasting blood glucose level of between 100 mg/dL and 125 mg/dl; bloodsugar level of between 140 mg/dL and 199 mg/dL two hours after ingestinga 75 gram glucose solution or a glucose solution of 1.75 grams ofglucose per kilogram of body weight, to a maximum dose of 75 grams;and/or glycated hemoglobin of between 5.7 percent and 6.4 percent.

The patient may exhibit one or more symptoms of diabetes, such asfasting blood glucose level greater than 125 mg/dl; blood sugar level ofat least 200 mg/dL two hours after ingesting a 75 gram glucose solutionor a glucose solution of 1.75 grams of glucose per kilogram of bodyweight, to a maximum dose of 75 grams; and/or glycated hemoglobin of atleast 6.5 percent.

The patient may have been diagnosed with type II diabetes.

The patient may be refractory to treatment with at least one compoundselected from the group consisting of: GLP-1, exenatide-1, exendin,exendin analog, exendin agonist, liraglutide, lixisenatide, albiglutide,exenatide LAR, a DPP-4 inhibitor, a GLP-1 receptor agonist, and anotherGLP-1 agonist; or such compound may be contraindicated foradministration to the patient.

The methods may further comprise administering to said patient ananti-diabetic agent or anti-obesity agent in addition to said modifiedFGF-21 polypeptide. Said anti-diabetic agent or anti-obesity agent maycomprise one or more of amylin, amylin agonist, sulfonylureas,calcitonin, glucagon, PPAR-gamma agonists, GPL-1 receptor agonists,dipeptidyl peptidase IV inhibitor, amylin analogs, biguanides, dopamineD2 receptor agonists, meglitinides, alpha-glucosidase inhibitor,antidyslipidemic bile acid sequestrant, exendin, exendin analog, exendinagonist, gastric inhibitory peptide (GIP), incretin peptide, insulin,SGLT2 inhibitor, a glucose reabsorption inhibitor, fenofibrate, fibrate,an anti-ghrelin antibody or antibody fragment, an fibroblast growthfactor receptor (FGFR)-1(IIIb), FGFR-1(IIIc), antibody or antibodyfragment, and/or FGFR-4(IIIc), an anti-CD38 antibody or antibodyfragment, an anti-MIC-1 antibody, or MIC-1 binding fragment, metforminor a combination of any of the foregoing.

In an exemplary embodiment, said anti-diabetic agent may be metformin,insulin glargine such as Lantus (Sanofi), sitagliptin such as Januvia,insulin aspart such as NovoLog and NovoRapid (Novo Nordisk), insulinlispro such as Humalog (Eli Lilly), liraglutide such as Victoza (NovoNordisk), insulin detemir such as Levemir (Novo Nordisk), sitagliptin incombination with metformin such as Janumet (Merck), soluble insulinaspart and protamine-crystallised insulin aspart in the ratio 30/70 suchas Novo Mix 30 (Novo Nordisk), or pioglitazone such as Actos (Takeda).

The method may be effective to cause weight loss.

The antidiabetic agents used in the combination with the modified FGF-21polypeptides of the present invention include, but are not limited to,insulin secretagogues or insulin sensitizers, MGAT2 inhibitors, or otherantidiabetic agents. These agents include, but are not limited to,dipeptidyl peptidase IV (DP4) inhibitors (for example, sitagliptin,saxagliptin, alogliptin, vildagliptin and the like), biguanides (forexample, metformin, phenformin and the like), sulfonyl ureas (forexample, glyburide, glimepiride, glipizide and the like), glucosidaseinhibitors (for example, acarbose, miglitol, and the like), PPARγagonists such as thiazolidinediones (for example, rosiglitazone,pioglitazone, and the like), PPAR α/γ dual agonists (for example,muraglitazar, tesaglitazar, aleglitazar, and the like), glucokinaseactivators (as described in Fyfe, M. C. T. et al., Drugs of the Future,34(8):641-653 (2009) and incorporated herein by reference), GPR40receptor modulators, GPR119 receptor modulators (MBX-2952, PSN821,APD597 and the like), SGLT2 inhibitors (dapagliflozin, canagliflozin,remagliflozin and the like), amylin analogs such as pramlintide, and/orinsulin. The modified FGF-21 polypeptides of the present invention mayalso be optionally employed in combination with agents for treatingcomplication of diabetes. These agents include PKC inhibitors and/or AGEinhibitors.

The modified FGF-21 polypeptides of the present invention may also beoptionally employed in combination with one or more hypophagic agentssuch as diethylpropion, phendimetrazine, phentermine, orlistat,sibutramine, lorcaserin, pramlintide, topiramate, MCHR1 receptorantagonists, oxyntomodulin, naltrexone, Amylin peptide, NPY Y5 receptormodulators, NPY Y2 receptor modulators, NPY Y4 receptor modulators,cetilistat, 5HT2c receptor modulators, and the like. The modified FGF-21polypeptides may also be employed in combination with an agonist of theglucagon-like peptide-1 receptor (GLP-1 R), such as exenatide,liraglutide, GPR-1(1-36) amide, GLP-1(7-36) amide, GLP-1(7-37) (asdisclosed in U.S. Pat. No. 5,614,492 to Habener, the disclosure of whichis incorporated herein by reference), which may be administered viainjection, intranasal, or by transdermal or buccal devices.

The modified FGF-21 polypeptides of the present invention may also beoptionally employed in combination with one or more other types oftherapeutic agents, such as DGAT inhibitors, LDL lowering drugs such asstatins (inhibitors of HMG CoA reductase) or inhibitors of cholesterolabsorption, modulators of PCSK9, drugs increase HDL such as CETPinhibitors.

In some embodiments, the non-naturally encoded amino acid may be linkedto a water soluble polymer. In some embodiments, the water solublepolymer comprises a poly(ethylene glycol) moiety. In some embodiments,the non-naturally encoded amino acid is linked to the water solublepolymer with a linker or is bonded to the water soluble polymer. In someembodiments, the poly(ethylene glycol) molecule is a bifunctionalpolymer. In some embodiments, the bifunctional polymer is linked to asecond polypeptide. In some embodiments, the second polypeptide is anumodified or modified FGF-21 polypeptide.

In some embodiments, the modified FGF-21 polypeptide comprises at leasttwo non-naturally encoded amino acids linked to a water soluble polymercomprising a poly(ethylene glycol) moiety. In some embodiments, one ormore non-naturally encoded amino acids are incorporated in one or moreof the following positions in modified FGF-21: before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminusof the protein) (amino acid positions corresponding to SEQ ID NO: 1 orthe corresponding amino acids in SEQ ID NOs: 2-7). In some embodiments,one or more non-naturally encoded amino acids are incorporated in one ormore of the following positions in modified FGF-21: 10, 52, 117, 126,131, 162, 87, 77, 83, 72, 69, 79, 91, 96, 108, and 110 (amino acidpositions corresponding to SEQ ID NO: 1 or the corresponding amino acidsof SEQ ID NOs: 2-7). In some embodiments, one or more non-naturallyencoded amino acids are incorporated in one or more of the followingpositions in modified FGF-21: 10, 52, 77, 117, 126, 131, 162 (amino acidpositions corresponding to SEQ ID NO: 1 or the corresponding amino acidsof SEQ ID NOs: 2-7). In some embodiments, one or more non-naturallyencoded amino acids are incorporated in one or more of the followingpositions in modified FGF-21: 87, 77, 83, 72 (amino acid positionscorresponding to SEQ ID NO: 1 or the corresponding amino acids of SEQ IDNOs: 2-7). In some embodiments, one or more non-naturally encoded aminoacids are incorporated in one or more of the following positions inmodified FGF-21: 69, 79, 91, 96, 108, and 110 (amino acid positionscorresponding to SEQ ID NO: 1 or the corresponding amino acids of SEQ IDNOs: 2-7). In some embodiments, one or more non-natural amino acids areincorporated in the leader or signal sequence of SEQ ID NOs: 3, 4, 6, 7,or other unmodified or modified FGF-21 sequence. In some embodiments,leader sequences may be chosen from SEQ ID NOs: 39, 40, 41, 42, 43, or44. In some embodiments, modified FGF-21 secretion constructs are clonedinto pVK7ara (Nde/Eco) with a leader sequences chosen from SEQ ID NOs:39, 40, 41, 42, 43, or 44.

In some embodiments, the non-naturally occurring amino acid at one ormore of these positions is linked to a water soluble polymer, includingbut not limited to, positions: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminus of theprotein) (amino acid positions corresponding to SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2-7). In some embodiments, thenon-naturally occurring amino acid at one or more positions from beforeposition 1 (i.e. at the N-terminus) through the C terminus in SEQ IDNOs: 34-36 is linked to a water soluble polymer. In some embodiments,the non-naturally occurring amino acid at one or more of these positionsis linked to a water soluble polymer, including but not limited to,positions: 10, 52, 117, 126, 131, 162, 87, 77, 83, 72, 69, 79, 91, 96,108, and 110 (amino acid positions corresponding to SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7). In some embodiments, thenon-naturally occurring amino acid at one or more of these positions islinked to a water soluble polymer, including but not limited to,positions: 10, 52, 77, 117, 126, 131, 162 (amino acid positionscorresponding to SEQ ID NO: 1 or the corresponding amino acids of SEQ IDNOs: 2-7). In some embodiments, the non-naturally occurring amino acidat one or more of these positions is linked to a water soluble polymer:87, 77, 83, 72 (amino acid positions corresponding to SEQ ID NO: 1 orthe corresponding amino acids of SEQ ID NOs: 2-7). In some embodiments,the non-naturally occurring amino acid at one or more of these positionsis linked to a water soluble polymer: 69, 79, 91, 96, 108, and 110(amino acid positions corresponding to SEQ ID NO: 1 or the correspondingamino acids of SEQ ID NOs: 2-7). In some embodiments, the one or morenon-naturally occurring amino acids in the leader or signal sequence ofSEQ ID NOs: 3, 4, 6, 7, 39, 40, 41, 42, 43, 44, or other modified FGF-21sequence is linked to a water soluble polymer. In some embodiments, theone or more non-naturally occurring amino acids in the leader or signalsequence of SEQ ID NOs: 3, 4, 6, 7, or other modified FGF-21 sequence islinked to a water soluble polymer.

In some embodiments, the modified FGF-21 polypeptide comprises at leastone substitution, addition or deletion that modulates affinity of themodified FGF-21 polypeptide for a FGF-21 polypeptide receptor or bindingpartner, including but not limited to, a protein, polypeptide, smallmolecule, or nucleic acid. In some embodiments, the modified FGF-21polypeptide comprises at least one substitution, addition, or deletionthat increases the stability of the modified FGF-21 polypeptide whencompared with the stability of the corresponding FGF-21 without the atleast one substitution, addition, or deletion, or when compared to acomparator compound such as the FGF-21 polypeptide of SEQ ID NO: 1, 201,or another unmodified or modified FGF-21 polypeptide. In someembodiments, the modified FGF-21 polypeptide comprises at least onesubstitution, addition, or deletion that decreases the immunogenicity ofthe modified FGF-21 polypeptide when compared with the immunogenicity ofthe corresponding FGF-21 without the at least one substitution,addition, or deletion, or when compared to a comparator compound such asthe FGF-21 polypeptide of SEQ ID NO: 1, 201, or another unmodified ormodified FGF-21 polypeptide. In some embodiments, the modified FGF-21polypeptide comprises at least one substitution, addition, or deletionthat increases serum half-life or circulation time of the modifiedFGF-21 polypeptide when compared with the serum half-life or circulationtime of the corresponding FGF-21 without the at least one substitution,addition, or deletion, or when compared to a comparator compound such asthe FGF-21 polypeptide of SEQ ID NO: 1, 201, or another unmodified ormodified FGF-21 polypeptide.

In some embodiments, the modified FGF-21 polypeptide comprises at leastone substitution, addition, or deletion that decreases deamidation ofthe modified FGF-21 polypeptide when compared to deamidation of thecorresponding FGF-21 without the at least one substitution, addition, ordeletion, or when compared to a comparator compound such as the FGF-21polypeptide of SEQ ID NO: 1, 201, or another unmodified or modifiedFGF-21 polypeptide. In some embodiments, the modified FGF-21 polypeptidecomprises at least one substitution, addition, or deletion thatincreases the aqueous solubility of the modified FGF-21 polypeptide whencompared to aqueous solubility of the corresponding FGF-21 without theat least one substitution, addition, or deletion, or when compared to acomparator compound such as the FGF-21 polypeptide of SEQ ID NO: 1,201,or another unmodified or modified FGF-21 polypeptide. In someembodiments, the modified FGF-21 polypeptide comprises at least onesubstitution, addition, or deletion that increases the solubility of themodified FGF-21 polypeptide produced in a host cell when compared to thesolubility of the corresponding FGF-21 without the at least onesubstitution, addition, or deletion, or when compared to a comparatorcompound such as the FGF-21 polypeptide of SEQ ID NO: 1, 201, or anotherunmodified or modified FGF-21 polypeptide. In some embodiments, themodified FGF-21 polypeptide comprises at least one substitution,addition, or deletion that increases the expression of the modifiedFGF-21 polypeptide in a host cell or increases synthesis in vitro whencompared to the expression or synthesis of the corresponding FGF-21without the at least one substitution, addition, or deletion, or whencompared to a comparator compound such as the FGF-21 polypeptide of SEQID NO: 1, 201, or another unmodified or modified FGF-21 polypeptide. Themodified FGF-21 polypeptide comprising this substitution may retainagonist activity and retains or improves expression levels in a hostcell. In some embodiments, the modified FGF-21 polypeptide comprises atleast one substitution, addition, or deletion that increases proteaseresistance or reduces protease cleavage (such as cleavage of C-terminalamino acids) of the modified FGF-21 polypeptide when compared to theprotease resistance of the corresponding FGF-21 without the at least onesubstitution, addition, or deletion, or when compared to a comparatorcompound such as the FGF-21 polypeptide of SEQ ID NO: 1,201, or anotherunmodified or modified FGF-21 polypeptide. U.S. Pat. No. 6,716,626indicated that potential sites that may be substituted to alter proteasecleavage include, but are not limited to, a monobasic site within 2residues of a proline. In some embodiments, the modified FGF-21polypeptide comprises at least one substitution, addition, or deletionthat modulates signal transduction activity of the modified FGF-21receptor when compared with the activity of the receptor uponinteraction with the corresponding FGF-21 polypeptide without the atleast one substitution, addition, or deletion, or when compared to acomparator compound such as the FGF-21 polypeptide of SEQ ID NO: 1, 201,or another unmodified or modified FGF-21 polypeptide. In someembodiments, the modified FGF-21 polypeptide comprises at least onesubstitution, addition, or deletion that modulates its binding toanother molecule such as a receptor when compared to the binding of thecorresponding FGF-21 polypeptide without the at least one substitution,addition, or deletion, or when compared to a comparator compound such asthe FGF-21 polypeptide of SEQ ID NO: 1, 201, or another unmodified ormodified FGF-21 polypeptide.

In some embodiments, the modified FGF-21 polypeptide comprises at leastone substitution, addition, or deletion that increases compatibility ofthe modified FGF-21 polypeptide with pharmaceutical preservatives (e.g.,m-cresol, phenol, benzyl alcohol) when compared to compatibility of thecorresponding FGF-21 without the at least one substitution, addition, ordeletion, or when compared to a comparator compound such as the FGF-21polypeptide of SEQ ID NO: 1, 201, or another unmodified or modifiedFGF-21 polypeptide. This increased compatibility would enable thepreparation of a preserved pharmaceutical formulation that maintains thephysiochemical properties and biological activity of the protein duringstorage. WO 2005/091944, which is incorporated by reference in itsentirety, discusses the following examples of FGF-21 muteins withenhanced pharmaceutical stability: the substitution with a chargedand/or polar but uncharged amino acid for one of the following: glycine42, glutamine 54, arginine 77, alanine 81, leucine 86, phenylalanine 88,lysine 122, histidine 125, arginine 126, proline 130, arginine 131,leucine 139, alanine 145, leucine 146, isoleucine 152, alanine 154,glutamine 156, glycine 161, serine 163, glycine 170, or serine 172 ofSEQ ID NO: 1 of WO 05/091944. A modified FGF-21 polypeptide of thepresent disclosure may include at least one of these substitutions atthe corresponding position in the polypeptide and/or may include one ormore other substitutions, additions, or deletions. In some embodiments,one or more non-natural amino acids are substituted at one or more ofthe following positions: glycine 42, glutamine 54, arginine 77, alanine81, leucine 86, phenylalanine 88, lysine 122, histidine 125, arginine126, proline 130, arginine 131, leucine 139, alanine 145,proline/leucine 146, isoleucine 152, alanine 154, glutamine 156, glycine161, serine 163, glycine 170, serine 172 (amino acid positionscorresponding to SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7). In some embodiments, one or more non-natural amino acids aresubstituted at one or more of the following positions: glutamate 91,arginine 131, glutamine 108, arginine 77, arginine 72, histidine 87,leucine 86, arginine 126, glutamate 110, tyrosine 83, proline 146,arginine 135, arginine 96, arginine 36, (amino acid positionscorresponding to SEQ ID NO: 1 or the corresponding amino acids in SEQ IDNOs: 2-7).

WO 05/091944 describes additional muteins of FGF-21 with enhancedpharmaceutical stability. Such muteins include the substitution of acysteine for two or more of the following in FGF-21 (see SEQ ID NO: 1 ofWO 05/091944): arginine 19, tyrosine 20, leucine 21, tyrosine 22,threonine 23, aspartate 24, aspartate 25, alanine 26, glutamine 27,glutamine 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine41, glycine 42, glycine 43, glutamate 50, glutamine 54, leucine 58,valine 62, leucine 66, glycine 67, lysine 69, arginine 72, phenylalanine73, glutamine 76, arginine 77, aspartate 79, glycine 80, alanine 81,leucine 82, glycine 84, serine 85, proline 90, alanine 92, serine 94,phenylalanine 95, leucine 100, aspartate 102, tyrosine 104, tyrosine107, serine 109, glutamate 110, proline 115, histidine 117, leucine 118,proline 119, asparagine 121, lysine 122, serine 123, proline 124,histidine 125, arginine 126, aspartate 127, alanine 129, proline 130,glycine 132, alanine 134, arginine 135, leucine 137, proline 138, orleucine 139. Modified FGF-21 polypeptides of the present disclosure mayinclude at least one of these substitutions at the correspondingposition in the polypeptide and/or may include one or more othersubstitutions, additions, or deletions.

WO 05/091944 further describes specific muteins of FGF-21 withengineered disulfide bonds (amino acids substituted with cysteine), inaddition to the naturally occurring one at Cys75-Cys93, are as follows:Gln76Cys-Ser109Cys, Cys75-Ser85Cys, Cys75-Ala92Cys, Phe73Cys-Cys93,Ser123Cys-His125Cys, Asp102Cys-Tyr104Cys, Asp127Cys-Gly132Cys,Ser94Cys-Glu110Cys, Pro115Cys-His117Cys, Asn121Cys-Asp127Cys,Leu100Cys-Asp102Cys, Phe95Cys-Tyr107Cys, Arg19CysPro138Cys,Tyr20Cys-Leu139Cys, Tyr22Cys-Leu137Cys, Arg77Cys-Asp79Cys,Pro90Cys-Ala92Cys, Glu50Cys-Lys69Cys, Thr23Cys-Asp25Cys,Ala31Cys-Gly43Cys, Gln28Cys-Gly43Cys, Thr23Cys-Gln28Cys,Val41Cys-Leu82Cys, Leu58Cys-Val62Cys, Gln54Cys-Leu66Cys,Ile35Cys-Gly67Cys, Gly67Cys-Arg72Cys, Ile35Cys-Gly84Cys,Arg72Cys-Gly84Cys, or Arg77Cys-Ala81Cys, where the numbering is based onSEQ ID NO: 1 of WO 05/091944. Additional muteins with engineereddisulfide bonds are Tyr22Cys-Leu139Cys; Asp24Cys-Arg135Cys;Leu118Cys-Gly132Cys; His117Cys-Pro130Cys; His117Cys-Ala129Cys;Leu82Cys-Pro119Cys; Gly80Cys-Ala129Cys; Gly43Cys-Pro124Cys;Gly42Cys-Arg126Cys; Gly42Cys-Pro124Cys; Gln28Cys-Pro124Cys;Gln27Cys-Ser123Cys; Ala26Cys-Lys122Cys; or Asp25Cys-Lys122Cys, where thenumbering is based on SEQ ID NO: 1 of WO 05/091944. Additional muteinswith engineered disulfide bonds are Leu118Cys-Ala134Cys;Leu21Cys-Leu33Cys; Ala26Cys-Lys122Cys;Leu21Cys-Leu33Cys/Leu118Cys-Ala134Cys, where the numbering is based onSEQ ID NO: 1 of WO 05/091944. Modified FGF-21 polypeptides of thepresent disclosure may include one or more of these substitutions at thecorresponding position(s) in the polypeptide and/or may include one ormore other substitutions, additions, or deletions.

WO 05/091944 describes additional muteins of FGF-21 that were PEGylated.These muteins had one of the following substitutions: D25C, D38C, L58C,K59C, P60C, K69C, D79C, H87C, E91C, E101C, D102C, L114C, L116C, K122C,R126C, P130C, P133C, P140C. WO 05/091944 describes cysteinesubstitutions at the following positions: 19, 21, 26, 28, 29, 30, 36,39, 42, 50, 56, 61, 64, 65, 68, 70, 71, 77, 81, 85, 86, 90, 92, 94, 98,107, 108, 112, 113, 123, and 124. WO 05/091944 indicates cysteinesubstitutions at the following positions: 24, 27, 37, 40, 44, 46, 49,57, 88, 89, 106, 110, 111, 115, 120, and 139. WO 05/091944 alsodescribes cysteine substitutions at the following positions: 18, 45, 47,48, 78, 83, 99, 103, 125, 128, 131, 132, and 138. WO 05/091944 alsodescribes cysteine substitutions at the following positions: 25, 38, 58,59, 60, 69, 79, 87, 91, 101, 102, 114, 116, 122, 126, 130, 133, and 140.

Modified FGF-21 polypeptides of the present disclosure may include oneor more of the aforementioned substitutions at the correspondingposition in the polypeptide and/or may include one or more othersubstitutions, additions, or deletions. Also provided herein arecompositions comprising a modified FGF-21 polypeptide adapted for use inthe methods described herein and a pharmaceutically acceptable carrieror excipient

In certain embodiments of the disclosure, the modified FGF-21polypeptide with at least one unnatural amino acid includes at least onepost-translational modification. In certain embodiments, thepost-translational modification is made in vitro. In certainembodiments, the post-translational modification is made in vivo in aeukaryotic cell or in a non-eukaryotic cell.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the protein includes atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is notnormally made by a non-eukaryotic cell. Examples of post-translationalmodifications include, but are not limited to, glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike. In certain embodiments, a protein or polypeptide of the disclosurecan comprise a secretion or localization sequence, an epitope tag, aFLAG tag, a polyhistidine tag, a GST fusion, and/or the like.

The disclosure also provides water soluble and hydrolytically stablederivatives of PEG derivatives and related hydrophilic polymers havingone or more acetylene or azide moieties. The PEG polymer derivativesthat contain acetylene moieties are highly selective for coupling withazide moieties that have been introduced selectively into proteins inresponse to a selector codon. Similarly, PEG polymer derivatives thatcontain azide moieties are highly selective for coupling with acetylenemoieties that have been introduced selectively into proteins in responseto a selector codon.

More specifically, the azide moieties comprise, but are not limited to,alkyl azides, aryl azides and derivatives of these azides. Thederivatives of the alkyl and aryl azides can include other substituentsso long as the acetylene-specific reactivity is maintained. Theacetylene moieties comprise alkyl and aryl acetylenes and derivatives ofeach. The derivatives of the alkyl and aryl acetylenes can include othersubstituents so long as the azide-specific reactivity is maintained.

The present disclosure provides conjugates of substances having a widevariety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a radionuclide; a cytotoxic compound; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin;an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; an actinicradiation excitable moiety; a photoisomerizable moiety; biotin; aderivative of biotin; a biotin analogue; a moiety incorporating a heavyatom; a chemically cleavable group; a photocleavable group; an elongatedside chain; a carbon-linked sugar; a redox-active agent; an aminothioacid; a toxic moiety; an isotopically labeled moiety; a biophysicalprobe; a phosphorescent group; a chemiluminescent group; an electrondense group; a magnetic group; an intercalating group; a chromophore; anenergy transfer agent; a biologically active agent; a detectable label;a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; aradiotransmitter; a neutron-capture agent; or any combination of theabove, or any other desirable compound or substance. The presentdisclosure also includes conjugates of substances having azide oracetylene moieties with PEG polymer derivatives having the correspondingacetylene or azide moieties. For example, a PEG polymer containing anazide moiety can be coupled to a biologically active molecule at aposition in the protein that contains a non-genetically encoded aminoacid bearing an acetylene functionality. The linkage by which the PEGand the biologically active molecule are coupled includes but is notlimited to the Huisgen [3+2] cycloaddition product.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group, an acetyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, or an alkynegroup.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group. In some embodiments, the non-naturally encoded aminoacid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises anaminooxy group. In some embodiments, the non-naturally encoded aminoacid comprises a hydrazide group. In some embodiments, the non-naturallyencoded amino acid comprises a hydrazine group. In some embodiments, thenon-naturally encoded amino acid residue comprises a semicarbazidegroup.

In some embodiments, the non-naturally encoded amino acid residuecomprises an azide group. In some embodiments, the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the non-naturally encoded amino acid comprises analkyne group. In some embodiments, the non-naturally encoded amino acidhas the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the modified FGF-21 polypeptide may be an agonist,partial agonist, antagonist, partial antagonist, or inverse agonist. Insome embodiments, the modified FGF-21 polypeptide comprises anon-naturally encoded amino acid linked to a water soluble polymer. Insome embodiments, the water soluble polymer comprises a poly(ethyleneglycol) moiety.

The present disclosure also provides isolated nucleic acids comprising apolynucleotide that hybridizes under stringent conditions to SEQ ID NO:8-14. The present disclosure also provides isolated nucleic acidscomprising a polynucleotide that hybridizes under stringent conditionsto SEQ ID NO: 8-14 wherein the polynucleotide comprises at least oneselector codon. The present disclosure also provides isolated nucleicacids comprising a polynucleotide that encodes the polypeptides shown asSEQ ID NOs: 1-7 or a modified FGF-21 polypeptide described herein. Insome embodiments, the isolated polyneucleotide described hereincomprises at least one selector codon, e.g., a selector codon thatencodes a non-naturally encoded amino acid contained in said modifiedFGF-21 polypeptide.

In some embodiments, the selector codon is selected from the groupconsisting of an amber codon, ochre codon, opal codon, a unique codon, arare codon, a five-base codon, and a four-base codon.

The present disclosure also provides methods of making a modified FGF-21polypeptide linked to a water soluble polymer. In some embodiments, themethod comprises contacting an isolated modified FGF-21 polypeptidecomprising a non-naturally encoded amino acid with a water solublepolymer comprising a moiety that reacts with the non-naturally encodedamino acid. In some embodiments, the non-naturally encoded amino acidincorporated into the modified FGF-21 polypeptide is reactive toward awater soluble polymer that is otherwise unreactive toward any of the 20common amino acids. In some embodiments, the non-naturally encoded aminoacid incorporated into the modified FGF-21 polypeptide is reactivetoward a linker, polymer, or biologically active molecule that isotherwise unreactive toward any of the 20 common amino acids.

In some embodiments, the modified FGF-21 polypeptide linked to the watersoluble polymer is made by reacting a modified FGF-21 polypeptidecomprising a carbonyl-containing amino acid with a linker, polymer, suchas poly(ethylene glycol) molecule, or biologically active molecule,comprising an aminooxy, hydrazine, hydrazide or semicarbazide group. Insome embodiments, the aminooxy, hydrazine, hydrazide or semicarbazidegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the modified FGF-21 polypeptide linked to the watersoluble polymer is made by reacting a poly(ethylene glycol) moleculecomprising a carbonyl group with a polypeptide comprising anon-naturally encoded amino acid that comprises an aminooxy, hydrazine,hydrazide or semicarbazide group.

In some embodiments, the modified FGF-21 polypeptide linked to the watersoluble polymer is made by reacting a modified FGF-21 polypeptidecomprising an alkyne-containing amino acid with a poly(ethylene glycol)molecule comprising an azide moiety. In some embodiments, the azide oralkyne group is linked to the poly(ethylene glycol) molecule through anamide linkage.

In some embodiments, the modified FGF-21 polypeptide linked to the watersoluble polymer is made by reacting a modified FGF-21 polypeptidecomprising an azide-containing amino acid with a poly(ethylene glycol)molecule comprising an alkyne moiety. In some embodiments, the azide oralkyne group is linked to the poly(ethylene glycol) molecule through anamide linkage.

In some embodiments, the water soluble polymer linked to the modifiedFGF-21 polypeptide comprises a polyalkylene glycol moiety. In someembodiments, the non-naturally encoded amino acid residue incorporatedinto the modified FGF-21 polypeptide comprises a carbonyl group, anaminooxy group, a hydrazide group, a hydrazine, a semicarbazide group,an azide group, or an alkyne group. In some embodiments, thenon-naturally encoded amino acid residue incorporated into the modifiedFGF-21 polypeptide comprises a carbonyl moiety and the water solublepolymer comprises an aminooxy, hydrazide, hydrazine, or semicarbazidemoiety. In some embodiments, the non-naturally encoded amino acidresidue incorporated into the modified FGF-21 polypeptide comprises analkyne moiety and the water soluble polymer comprises an azide moiety.In some embodiments, the non-naturally encoded amino acid residueincorporated into the modified FGF-21 polypeptide comprises an azidemoiety and the water soluble polymer comprises an alkyne moiety.

The present disclosure also provides cells comprising a polynucleotideencoding the modified FGF-21 polypeptide comprising a selector codon. Insome embodiments, the cells comprise an orthogonal RNA synthetase and/oran orthogonal tRNA for substituting a non-naturally encoded amino acidinto the modified FGF-21 polypeptide.

The present disclosure also provides methods of making a modified FGF-21polypeptide comprising a non-naturally encoded amino acid. In someembodiments, the methods comprise culturing cells comprising apolynucleotide or polynucleotides encoding a modified FGF-21polypeptide, an orthogonal RNA synthetase and/or an orthogonal tRNAunder conditions to permit expression of the modified FGF-21polypeptide; and purifying the modified FGF-21 polypeptide from thecells and/or culture medium.

Included within the scope of this disclosure is the FGF-21 leader orsignal sequence joined to a modified FGF-21 coding region, as well as aheterologous signal sequence joined to a modified FGF-21 coding region.The heterologous leader or signal sequence selected should be one thatis recognized and processed, e.g. by host cell secretion system tosecrete and possibly cleaved by a signal peptidase, by the host cell.Leader sequences of the present disclosure may be chosen from thefollowing: the three leucine leader from SEQ ID NO: 3 and SEQ ID NO: 6(amino acid positions 1-28), the two leucine leader from SEQ ID NO: 4and SEQ ID NO: 7 (amino acid positions 1-27), the His tag from SEQ IDNO: 2 (amino acid positions 1-10), SEQ ID NO: 39, SEQ ID NO: 40, SEQ IDNO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44. A method oftreating a condition or disorder with the modified FGF-21 of the presentdisclosure is meant to imply treating with a modified FGF-21 polypeptidewith or without a signal or leader peptide.

The present disclosure also provides methods of inducing an increase inglucose uptake in adipocyte cells, said method comprising administeringmodified FGF-21 to said cells in an amount effective to induce anincrease in glucose uptake. Said increase in glucose uptake may cause anincrease in energy expenditure by faster and more efficient glucoseutilization.

II. General Recombinant Nucleic Acids and Methods for Use with theDisclosed Modified FGF-21 Polypeptides

In numerous embodiments of the present disclosure, nucleic acidsencoding a modified FGF-21 polypeptide of interest may be isolated,cloned and often altered using recombinant methods. Such embodiments maybe used, including but not limited to, for protein expression or duringthe generation of variants, derivatives, expression cassettes, or othersequences derived from a modified FGF-21 polypeptide. In someembodiments, the sequences encoding the modified FGF-21 polypeptides ofthe disclosure are operably linked to a heterologous promoter. In someembodiments DNA codon usage in the polynucleotide sequences encoding themodified FGF-21 polpeptide may be optimized for E. coli or a mammaliancell (e.g. CHO) expression using techniques that are well known in theart

An exemplary cDNA encoding the P-form of FGF-21 without the leadersequence is shown as SEQ ID NO: 8. This polypeptide is shown as SEQ IDNO: 1.

An exemplary cDNA encoding a His tagged P-form of FGF-21 without aleader sequence is shown as SEQ ID NO: 9. This polypeptide is shown asSEQ ID NO: 2.

An exemplary cDNA encoding the P-form of FGF-21 with a leader sequencecontaining 3 leucines together is shown as SEQ ID NO: 10. Thispolypeptide is shown as SEQ ID NO: 3.

An exemplary cDNA encoding the P-form of FGF-21 with a leader sequencecontaining 2 leucines together is shown as SEQ ID NO: 11. Thispolypeptide is shown as SEQ ID NO: 4.

An exemplary cDNA encoding the L-form of FGF-21 without the leadersequence is shown as SEQ ID NO: 12. This polypeptide is shown as SEQ IDNO: 5.

An exemplary cDNA encoding the L-form of FGF-21 with a leader sequencecontaining 3 leucines together is shown as SEQ ID NO: 13. Thispolypeptide is shown as SEQ ID NO: 6.

An exemplary cDNA encoding the L-form of FGF-21 with a leader sequencecontaining 2 leucines together is shown as SEQ ID NO: 14. Thispolypeptide is shown as SEQ ID NO: 7.

A nucleotide sequence encoding a modified FGF-21 polypeptide describedherein may be synthesized on the basis of the amino acid sequence of theparent polypeptide, including but not limited to, having the amino acidsequence shown in SEQ ID NO: 1-7 and then changing the nucleotidesequence so as to effect introduction (i.e., incorporation orsubstitution) or removal (i.e., deletion or substitution) of therelevant amino acid residue(s). The nucleotide sequence may beconveniently modified by site-directed mutagenesis in accordance withconventional methods. Alternatively, the nucleotide sequence may beprepared by chemical synthesis, including but not limited to, by usingan oligonucleotide synthesizer, wherein oligonucleotides are designedbased on the amino acid sequence of the desired polypeptide, andpreferably selecting those codons that are favored in the host cell inwhich the recombinant polypeptide is to be produced. For example,several small oligonucleotides coding for portions of the desiredpolypeptide may be synthesized and assembled by PCR, ligation orligation chain reaction. See, e.g., Barany, et al., Proc. Natl. Acad.Sci. 88: 189-193 (1991); U.S. Pat. No. 6,521,427 which are incorporatedby reference herein.

The disclosure also relates to eukaryotic host cells, non-eukaryotichost cells, and organisms for the in vivo incorporation of an unnaturalamino acid via orthogonal tRNA/RS pairs. Host cells are geneticallyengineered (including but not limited to, transformed, transduced ortransfected) with the polynucleotides of the disclosure or constructswhich include a polynucleotide of the disclosure, including but notlimited to, a vector of the disclosure, which can be, for example, acloning vector or an expression vector. For example, the coding regionsfor the orthogonal tRNA, the orthogonal tRNA synthetase, and the proteinto be derivatized are operably linked to gene expression controlelements that are functional in the desired host cell. The vector canbe, for example, in the form of a plasmid, a cosmid, a phage, abacterium, a virus, a naked polynucleotide, or a conjugatedpolynucleotide. The vectors may be introduced into cells and/ormicroorganisms by standard methods.

Selector Codons

Selector codons of the disclosure expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,but is not limited to, a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), anochre codon, or an opal codon (UGA), an unnatural codon, a four or morebase codon, a rare codon, or the like. It is readily apparent to thoseof ordinary skill in the art that there is a wide range in the number ofselector codons that can be introduced into a desired gene orpolynucleotide, including but not limited to, one or more, two or more,three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotideencoding at least a portion of the modified FGF-21 polypeptide.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of one or more unnatural (i.e.,non-naturally encoded) amino acids in vivo. For example, an O-tRNA isproduced that recognizes the stop codon, including but not limited to,UAG, and is aminoacylated by an O-RS with a desired unnatural aminoacid. This O-tRNA is not recognized by the naturally occurring host'saminoacyl-tRNA synthetases. Conventional site-directed mutagenesis canbe used to introduce the stop codon, including but not limited to, TAG,at the site of interest in a polypeptide of interest. See, e.g., Sayers,J. R., et al. (1988), 5′-3′ Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis. Nucleic Acids Res, 16:791-802.When the O-RS, O-tRNA and the nucleic acid that encodes the polypeptideof interest are combined in vivo, the unnatural amino acid isincorporated in response to the UAG codon to give a polypeptidecontaining the unnatural amino acid at the specified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurring tRNAArg,which exists as a minor species in Escherichia coli. Some organisms donot use all triplet codons. An unassigned codon AGA in Micrococcusluteus has been utilized for insertion of amino acids in an in vitrotranscription/translation extract. See, e.g., Kowal and Oliver, Nucl.Acid. Res., 25:4685 (1997). Components of the present disclosure can begenerated to use these rare codons in vivo.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, but are not limited to,AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the disclosure includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, including but not limited to, one or multiple unnatural aminoacids into the same protein. For example, in the presence of mutatedO-tRNAs, including but not limited to, a special frameshift suppressortRNAs, with anticodon loops, for example, with at least 8-10 ntanticodon loops, the four or more base codon is read as single aminoacid. In other embodiments, the anticodon loops can decode, includingbut not limited to, at least a four-base codon, at least a five-basecodon, or at least a six-base codon or more. Since there are 256possible four-base codons, multiple unnatural amino acids can be encodedin the same cell using a four or more base codon.

In one embodiment, extended codons based on rare codons or nonsensecodons can be used in the present disclosure, which can reduce missensereadthrough and frameshift suppression at other unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002), Nature Biotechnology, 20:177-182. See, also, Wu, Y., et al.,(2002) J. Am. Chem. Soc. 124:14626-14630.

Genes coding for proteins or polypeptides of interest such as a modifiedFGF-21 polypeptide can be mutagenized using methods known to one ofordinary skill in the art and described herein to include, for example,one or more selector codon for the incorporation of an unnatural aminoacid. For example, a nucleic acid for a protein of interest ismutagenized to include one or more selector codon, providing for theincorporation of one or more unnatural amino acids. The disclosureincludes any such variant, including but not limited to, mutant,versions of any protein, for example, including at least one unnaturalamino acid.

Nucleic acid molecules encoding a protein of interest such as a modifiedFGF-21 polypeptide may be readily mutated to introduce a cysteine at anydesired position of the polypeptide. Cysteine is widely used tointroduce reactive molecules, water soluble polymers, proteins, or awide variety of other molecules, onto a protein of interest.

III. Non-Naturally Encoded Amino Acids

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present disclosure. Any number of non-naturally encodedamino acids can be introduced into a modified FGF-21 polypeptide. Ingeneral, the introduced non-naturally encoded amino acids aresubstantially chemically inert toward the 20 common, genetically-encodedamino acids (i.e., alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine). In some embodiments, thenon-naturally encoded amino acids include side chain functional groupsthat react efficiently and selectively with functional groups not foundin the 20 common amino acids (including but not limited to, azido,ketone, aldehyde and aminooxy groups) to form stable conjugates. Forexample, a modified FGF-21 polypeptide that includes a non-naturallyencoded amino acid containing an azido functional group can be reactedwith a polymer (including but not limited to, poly(ethylene glycol) or,alternatively, a second polypeptide containing an alkyne moiety to forma stable conjugate resulting for the selective reaction of the azide andthe alkyne functional groups to form a Huisgen [3+2] cycloadditionproduct.

The generic structure of an alpha-amino acid is illustrated as follows(Formula I):

A non-naturally encoded amino acid is typically any structure having theabove-listed formula wherein the R group is any substituent other thanone used in the twenty natural amino acids, and may be suitable for usein the modified FGF-21 polypeptides of the present disclosure. Becausethe non-naturally encoded amino acids of the disclosure typically differfrom the natural amino acids only in the structure of the side chain,the non-naturally encoded amino acids form amide bonds with other aminoacids, including but not limited to, natural or non-naturally encoded,in the same manner in which they are formed in naturally occurringpolypeptides. However, the non-naturally encoded amino acids have sidechain groups that distinguish them from the natural amino acids. Forexample, R optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-,hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether,thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid,hydroxylamine, amino group, or the like or any combination thereof.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present disclosure and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of ordinary skill in the art. Fororganic synthesis techniques, see, e.g., Organic Chemistry by Fessendonand Fessendon, (1982, Second Edition, Willard Grant Press, BostonMass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wileyand Sons, New York); and Advanced Organic Chemistry by Carey andSundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).See, also, U.S. Pat. Nos. 7,045,337 and 7,083,970, which areincorporated by reference herein. In addition to unnatural amino acidsthat contain novel side chains, unnatural amino acids that may besuitable for use in the present disclosure also optionally comprisemodified backbone structures, including but not limited to, asillustrated by the structures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the disclosure optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like, and are suitable foruse in the present disclosure. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C₆-C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present disclosure include, but arenot limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentdisclosure include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenylalanines, and meta-substitutedphenylalanines, where the substituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present disclosure include, but are not limitedto, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike.

In one embodiment, compositions of a modified FGF-21 polypeptidecomprising an unnatural amino acid (such asp-(propargyloxy)-phenylalanine) are provided. Various compositionscomprising p-(propargyloxy)-phenylalanine and, including but not limitedto, proteins and/or cells, are also provided. In one aspect, acomposition that includes the p-(propargyloxy)-phenylalanine unnaturalamino acid, further includes an orthogonal tRNA. The unnatural aminoacid can be bonded (including but not limited to, covalently) to theorthogonal tRNA, including but not limited to, covalently bonded to theorthogonal tRNA though an amino-acyl bond, covalently bonded to a 3′OHor a 2′OH of a terminal ribose sugar of the orthogonal tRNA, etc.

The modified FGF-21 polypeptide described herein may comprise anon-naturally encoded amino acid described in U.S. Pat. No. 8,012,931,which is incorporated herein by reference in its entirety.

Structure and Synthesis of Non-Natural Amino Acids: Carbonyl,Carbonyl-Like, Masked Carbonyl, Protected Carbonyl Groups, andHydroxylamine Groups

In some embodiments the present disclosure provides modified FGF-21linked to a water soluble polymer, e.g., a PEG, by an oxime bond. Manytypes of non-naturally encoded amino acids are suitable for formation ofoxime bonds. These include, but are not limited to, non-naturallyencoded amino acids containing a carbonyl, dicarbonyl, carbonyl-like,masked carbonyl, protected carbonyl, or hydroxylamine group. Such aminoacids, their structure and synthesis are described in U.S. Pat. No.8,012,931, which is incorporated herein by reference in its entirety.

IV. Structure and Synthesis of Non-Natural Amino Acids:Hydroxylamine-Containing Amino Acids

U.S. Provisional Patent Application No. 60/638,418 is incorporated byreference in its entirety. Thus, the disclosures provided in Section V(entitled “Non-natural Amino Acids”), Part B (entitled “Structure andSynthesis of Non-Natural Amino Acids: Hydroxylamine-Containing AminoAcids”), in U.S. Provisional Patent Application No. 60/638,418 applyfully to the methods, compositions, techniques and strategies formaking, purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein to the same extent as if such disclosureswere fully presented herein. U.S. Patent Publication Nos. 2006/0194256,2006/0217532, and 2006/0217289 and WO 2006/069246 entitled “Compositionscontaining, methods involving, and uses of non-natural amino acids andpolypeptides,” are also incorporated herein by reference in theirentirety.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids suitable for use in the presentdisclosure are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of ordinary skillin the art. For organic synthesis techniques, see, e.g., OrganicChemistry by Fessendon and Fessendon, (1982, Second Edition, WillardGrant Press, Boston Mass.); Advanced Organic Chemistry by March (ThirdEdition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistryby Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press,New York). Additional publications describing the synthesis of unnaturalamino acids include, e.g., WO 2002/085923 entitled “In vivoincorporation of Unnatural Amino Acids;” Matsoukas et al., (1995) J.Med. Chem., 38, 4660-4669; King, F. E. & Kidd, D. A. A. (1949) A NewSynthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates. J. Chem. Soc., 3315-3319; Friedman, O. M. &Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752;Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of7-Chloro-4[[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis ofL-and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid andAppropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and,Subasinghe et al., (1992) Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Seealso, U.S. Patent Publication No. U.S. 2004/0198637 entitled “ProteinArrays,” which is incorporated by reference herein.

A. Carbonyl Reactive Groups

Amino acids with a carbonyl reactive group allow for a variety ofreactions to link molecules (including but not limited to, PEG or otherwater soluble molecules) via nucleophilic addition or aldol condensationreactions among others.

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein. Other carbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art.

In some embodiments, a modified FGF-21 polypeptide comprising anon-naturally encoded amino acid may be chemically modified to generatea reactive carbonyl functional group. For instance, an aldehydefunctionality useful for conjugation reactions can be generated from afunctionality having adjacent amino and hydroxyl groups. Where thebiologically active molecule is a polypeptide, for example, anN-terminal serine or threonine (which may be normally present or may beexposed via chemical or enzymatic digestion) can be used to generate analdehyde functionality under mild oxidative cleavage conditions usingperiodate. See, e.g., Gaertner, et al., Bioconjug. Chem. 3: 262-268(1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992);Gaertner et al., J. Biol. Chem. 269:7224-7230 (1994). However, methodsknown in the art are restricted to the amino acid at the N-terminus ofthe peptide or protein.

In the present disclosure, a non-naturally encoded amino acid bearingadjacent hydroxyl and amino groups can be incorporated into thepolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which isincorporated by reference herein.

The carbonyl functionality can be reacted selectively with a hydrazine-,hydrazide-, hydroxylamine-, or semicarbazide-containing reagent undermild conditions in aqueous solution to form the corresponding hydrazone,oxime, or semicarbazone linkages, respectively, that are stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonylgroup allows for selective modification in the presence of the otheramino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem.Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug.Chem. 3:138-146 (1992); Mahal, L. K., et al., Science 276:1125-1128(1997).

B. Hydrazine, Hydrazide or Semicarbazide Reactive Groups

Non-naturally encoded amino acids containing a nucleophilic group, suchas a hydrazine, hydrazide or semicarbazide, allow for reaction with avariety of electrophilic groups to form conjugates (including but notlimited to, with PEG or other water soluble polymers).

Hydrazide-, hydrazine-, and semicarbazide-containing amino acids areavailable from commercial sources. For instance, L-glutamate-γ-hydrazideis available from Sigma Chemical (St. Louis, Mo.). Other amino acids notavailable commercially can be prepared by one of ordinary skill in theart. See, e.g., U.S. Pat. No. 6,281,211, which is incorporated byreference herein.

Modified FGF-21 polypeptides containing non-naturally encoded aminoacids that bear hydrazide, hydrazine or semicarbazide functionalitiescan be reacted efficiently and selectively with a variety of moleculesthat contain aldehydes or other functional groups with similar chemicalreactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995). The unique reactivity of hydrazide, hydrazine andsemicarbazide functional groups makes them significantly more reactivetoward aldehydes, ketones and other electrophilic groups as compared tothe nucleophilic groups present on the 20 common amino acids (includingbut not limited to, the hydroxyl group of serine or threonine or theamino groups of lysine and the N-terminus).

C. Aminooxy-Containing Amino Acids

Non-naturally encoded amino acids containing an aminooxy (also called ahydroxylamine) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl-containing group suchas a ketone.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G., Life Sci.60: 1635-1641 (1997). Other aminooxy-containing amino acids can beprepared by one of ordinary skill in the art.

D. Azide and Alkyne Reactive Groups

The unique reactivity of azide and alkyne functional groups makes themextremely useful for the selective modification of polypeptides andother biological molecules. Organic azides, particularly alphaticazides, and alkynes are generally stable toward common reactive chemicalconditions. In particular, both the azide and the alkyne functionalgroups are inert toward the side chains (i.e., R groups) of the 20common amino acids found in naturally-occurring polypeptides. Whenbrought into close proximity, however, the “spring-loaded” nature of theazide and alkyne groups is revealed and they react selectively andefficiently via Huisgen [3+2] cycloaddition reaction to generate thecorresponding triazole. See, e.g., Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin,J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction involves a selectivecycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANICSYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R.in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)rather than a nucleophilic substitution, the incorporation ofnon-naturally encoded amino acids bearing azide and alkyne-containingside chains permits the resultant polypeptides to be modifiedselectively at the position of the non-naturally encoded amino acid.Cycloaddition reaction involving azide or alkyne-containing modifiedFGF-21 polypeptide can be carried out at room temperature under aqueousconditions by the addition of Cu(II) (including but not limited to, inthe form of a catalytic amount of CuSO₄) in the presence of a reducingagent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See,e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe,C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al.,Angew. Chem. Int. Ed. 41:2596-2599 (2002). Exemplary reducing agentsinclude, including but not limited to, ascorbate, metallic copper,quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, andan applied electric potential.

In some cases, where a Huisgen [3+2] cycloaddition reaction between anazide and an alkyne is desired, the modified FGF-21 polypeptide maycomprise a non-naturally encoded amino acid comprising an alkyne moietyand the water soluble polymer to be attached to the amino acid maycomprise an azide moiety. Alternatively, the converse reaction (i.e.,with the azide moiety on the amino acid and the alkyne moiety present onthe water soluble polymer) can also be performed.

The azide functional group can also be reacted selectively with a watersoluble polymer containing an aryl ester and appropriatelyfunctionalized with an aryl phosphine moiety to generate an amidelinkage. The aryl phosphine group reduces the azide in situ and theresulting amine then reacts efficiently with a proximal ester linkage togenerate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi,Science 287, 2007-2010 (2000). The azide-containing amino acid can beeither an alkyl azide (including but not limited to,2-amino-6-azido-1-hexanoic acid) or an aryl azide(p-azido-phenylalanine).

The azide functional group can also be reacted selectively with a watersoluble polymer containing a thioester and appropriately functionalizedwith an aryl phosphine moiety to generate an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine thenreacts efficiently with the thioester linkage to generate thecorresponding amide.

Alkyne-containing amino acids are commercially available. For example,propargylglycine is commercially available from Peptech (Burlington,Mass.). Alternatively, alkyne-containing amino acids can be preparedaccording to standard methods. For instance, p-propargyloxyphenylalaninecan be synthesized, for example, as described in Deiters, A., et al., J.Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalaninecan be synthesized as described in Kayser, B., et al., Tetrahedron53(7): 2475-2484 (1997). Other alkyne-containing amino acids can beprepared by one of ordinary skill in the art.

Azide-containing amino acids are available from commercial sources. Forinstance, 4-azidophenylalanine can be obtained from Chem-ImpexInternational, Inc. (Wood Dale, Ill.). For those azide-containing aminoacids that are not commercially available, the azide group can beprepared relatively readily using standard methods known to those ofordinary skill in the art, including but not limited to, viadisplacement of a suitable leaving group (including but not limited to,halide, mesylate, tosylate) or via opening of a suitably protectedlactone. See, e.g., Advanced Organic Chemistry by March (Third Edition,1985, Wiley and Sons, New York).

A molecule that can be added to a protein of the disclosure through a[3+2] cycloaddition includes virtually any molecule with an azide oralkynyl derivative. Molecules include, but are not limited to, dyes,fluorophores, crosslinking agents, saccharide derivatives, polymers(including but not limited to, polymers comprising polyethylene glycol),photocrosslinkers, cytotoxic compounds, affinity labels, derivatives ofbiotin, resins, beads, a second protein or polypeptide (or more),polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metalchelators, cofactors, fatty acids, carbohydrates, and the like. Thesemolecules can be added to an unnatural amino acid with an alkynyl group,including but not limited to, p-propargyloxyphenylalanine, or azidogroup, including but not limited to, p-azido-phenylalanine,respectively.

E. Aminothiol Reactive Groups

The unique reactivity of beta-substituted aminothiol functional groupsmakes them extremely useful for the selective modification ofpolypeptides and other biological molecules that contain aldehyde groupsvia formation of the thiazolidine. See, e.g., J. Shao and J. Tam, J. Am.Chem. Soc. 1995, 117 (14) 3893-3899. In some embodiments,beta-substituted aminothiol amino acids can be incorporated intomodified FGF-21 polypeptides and then reacted with water solublepolymers comprising an aldehyde functionality. In some embodiments, awater soluble polymer, drug conjugate or other payload can be coupled toa modified FGF-21 polypeptide comprising a beta-substituted aminothiolamino acid via formation of the thiazolidine.

F. Additional Reactive Groups

Additional reactive groups and non-naturally encoded amino acids thatcan be incorporated into modified FGF-21 polypeptides of the disclosureare described in the following patent applications which are allincorporated by reference in their entirety herein: U.S. PatentPublication No. 2006/0194256, U.S. Patent Publication No. 2006/0217532,U.S. Patent Publication No. 2006/0217289, U.S. Provisional Patent No.60/755,338; U.S. Provisional Patent No. 60/755,711; U.S. ProvisionalPatent No. 60/755,018; International Patent Application No.PCT/US06/49397; WO 2006/069246; U.S. Provisional Patent No. 60/743,041;U.S. Provisional Patent No. 60/743,040; International Patent ApplicationNo. PCT/US06/47822; U.S. Provisional Patent No. 60/882,819; U.S.Provisional Patent No. 60/882,500; and U.S. Provisional Patent No.60/870,594.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, includingbut not limited to, for incorporation into a protein. For example, thehigh charge density of α-amino acids suggests that these compounds areunlikely to be cell permeable. Natural amino acids are taken up into theeukaryotic cell via a collection of protein-based transport systems. Arapid screen can be done which assesses which unnatural amino acids, ifany, are taken up by cells. See, e.g., the toxicity assays in, e.g.,U.S. Patent Publication No. U.S. 2004/0198637 entitled “Protein Arrays”which is incorporated by reference herein; and Liu, D. R. & Schultz, P.G. (1999) Progress toward the evolution of an organism with an expandedgenetic code. PNAS United States 96:4780-4785. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a cell, the disclosure provides such methods. Forexample, biosynthetic pathways for unnatural amino acids are optionallygenerated in host cell by adding new enzymes or modifying existing hostcell pathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a eukaryotic cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, for example, in GenBank. Artificially evolvedenzymes are also optionally added into a cell in the same manner. Inthis manner, the cellular machinery and resources of a cell aremanipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the World Wide Web atmaxygen.com), is optionally used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™, developed by Genencor (available on the WorldWide Web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, those identified through functionalgenomics, and molecular evolution and design. Diversa Corporation(available on the World Wide Web at diversa.com) also providestechnology for rapidly screening libraries of genes and gene pathways,including but not limited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the disclosure is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

V. In Vivo Generation of Modified FGF-21 Polypeptides ComprisingNon-Naturally-Encoded Amino Acids

The modified FGF-21 polypeptides of the disclosure can be generated invivo using modified tRNA and tRNA synthetases to add to or substituteamino acids that are not encoded in naturally-occurring systems. Suchmethods are described in U.S. Pat. No. 8,012,931, which is incorporatedherein by reference in its entirety.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Pat. Nos. 7,045,337 and 7,083,970 which are incorporated byreference herein. These methods involve generating a translationalmachinery that functions independently of the synthetases and tRNAsendogenous to the translation system (and are therefore sometimesreferred to as “orthogonal”). Typically, the translation systemcomprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNAsynthetase (O-RS). Typically, the O-RS preferentially aminoacylates theO-tRNA with at least one non-naturally occurring amino acid in thetranslation system and the O-tRNA recognizes at least one selector codonthat is not recognized by other tRNAs in the system. The translationsystem thus inserts the non-naturally-encoded amino acid into a proteinproduced in the system, in response to an encoded selector codon,thereby “substituting” an amino acid into a position in the encodedpolypeptide.

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the modified FGF-21 polynucleotide coding sequence using mutagenesismethods known in the art (including but not limited to, site-specificmutagenesis, cassette mutagenesis, restriction selection mutagenesis,etc.).

VI. Location of Non-Naturally Encoded Amino Acids in Modified FGF-21Polypeptides

The present disclosure contemplates incorporation of one or morenon-naturallyencoded amino acids into modified FGF-21 polypeptides. Oneor more non-naturally encoded amino acids may be incorporated at aparticular position which does not disrupt activity of the polypeptide.This can be achieved by making “conservative” substitutions, includingbut not limited to, substituting hydrophobic amino acids withhydrophobic amino acids, bulky amino acids for bulky amino acids,hydrophilic amino acids for hydrophilic amino acids and/or inserting thenon-naturally-occurring amino acid in a location that is not requiredfor activity.

In some embodiments, the modified FGF-21 polypeptides of the disclosurecomprise one or more non-naturally occurring amino acids positioned in aregion of the protein that does not disrupt the structure of thepolypeptide.

In some embodiments, the amino acid sequence in the modified FGF-21polypeptide described herein may comprise at least one non-naturallyencoded amino acid. The at least one non-naturally encoded amino acidsmay be incorporated at any positions in the amino acid sequence,including positions corresponding to amino acids 1-181 from SEQ ID NO: 1or before position 1 (i.e. at the N-terminus) or at position 182 (i.e.,at the carboxyl terminus of the protein) therein or the correspondingamino acids in SEQ ID NOs: 2-7 or another modified FGF-21 polypeptide ofthe disclosure. In some embodiments, the at least one non-naturallyencoded amino acid may be at a position corresponding to amino acid 10,36, 52, 117, 126, 131, 135, 146, 162, 87, 77, 83, 72, 69, 79, 91, 96,108, or 110 of SEQ ID NO: 1. In some embodiments, the at least onenon-naturally encoded amino acid may be at a position corresponding toamino acid 72, 77, 86, 87, 91, 108, 110, 126, 131, or 146 of SEQ IDNO: 1. In some embodiments, the at least one non-naturally occurringamino acid may be at a position corresponding to amino acid 108 of SEQID NO: 1. In some embodiments, the at least one non-naturally encodedamino acid may be a phenylalanine derivative. In some embodiments, theposition corresponding to amino acid 108 of SEQ ID NO: 1 may be aphenylalanine derivative. In some embodiments, the at least onenon-naturally encoded amino acid may be para-acetyl-L-phenylalanine. Insome embodiments, the position corresponding to amino acid 108 of SEQ IDNO: 1 may be para-acetyl-L-phenylalanine. In one embodiment, thenon-naturally occurring amino acid is at the 91 position in FGF-21 (SEQID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). In oneembodiment, the non-naturally occurring amino acid is at the 131position in FGF-21 (SEQ ID NO: 1 or the corresponding amino acids of SEQID NOs: 2-7). In one embodiment, the non-naturally occurring amino acidis at the 108 position in FGF-21 (SEQ ID NO: 1 or the correspondingamino acids of SEQ ID NOs: 2-7). In one embodiment, the non-naturallyoccurring amino acid is at the 77 position in FGF-21 (SEQ ID NO: 1 orthe corresponding amino acids of SEQ ID NOs: 2-7). In one embodiment,the non-naturally occurring amino acid is at the 72 position in FGF-21(SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). Inone embodiment, the non-naturally occurring amino acid is at the 87position in FGF-21 (SEQ ID NO: 1 or the corresponding amino acids of SEQID NOs: 2-7). In one embodiment, the non-naturally occurring amino acidis at the 86 position in FGF-21 (SEQ ID NO: 1 or the corresponding aminoacids of SEQ ID NOs: 2-7). In one embodiment, the non-naturallyoccurring amino acid is at the 126 position in FGF-21 (SEQ ID NO: 1 orthe corresponding amino acids of SEQ ID NOs: 2-7). In one embodiment,the non-naturally occurring amino acid is at the 110 position in FGF-21(SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). Inone embodiment, the non-naturally occurring amino acid is at the 83position in FGF-21 (SEQ ID NO: 1 or the corresponding amino acids of SEQID NOs: 2-7). In one embodiment, the non-naturally occurring amino acidis at the 146 position in FGF-21 (SEQ ID NO: 1 or the correspondingamino acids of SEQ ID NOs: 2-7). In one embodiment, the non-naturallyoccurring amino acid is at the 135 position in FGF-21 (SEQ ID NO: 1 orthe corresponding amino acids of SEQ ID NOs: 2-7). In one embodiment,the non-naturally occurring amino acid is at the 96 position in FGF-21(SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7). Inone embodiment, the non-naturally occurring amino acid is at the 36position in FGF-21 (SEQ ID NO: 1 or the corresponding amino acids of SEQID NOs: 2-7).

In another embodiment, the amino acid sequence in the modified FGF-21polypeptide described herein may comprise a non-naturally encoded aminoacid at a position corresponding to amino acid 108 of SEQ ID NO: 1 andat least one other non-naturally encoded amino acid at a positioncorresponding to any other one of amino acids 1-181 from SEQ ID NO: 1 orbefore position 1 (i.e. at the N-terminus) or at position 182 (i.e., atthe carboxyl terminus of the protein) therein or the corresponding aminoacids in SEQ ID NOs: 2-7 or another modified FGF-21 polypeptide of thedisclosure. In another embodiment, the at least one other non-naturallyencoded amino acid may be at a position corresponding to amino acid 10,36, 52, 117, 126, 131, 135, 146, 162, 87, 77, 83, 72, 69, 79, 91, 96,108, or 110 of SEQ ID NO: 1. In another embodiment, the at least oneother non-naturally encoded amino acid may be at a positioncorresponding to amino acid 91 or 131 of SEQ ID NO: 1.

In another embodiment, there is a non-naturally occurring amino acid at91 and one or more other non-naturally occurring amino acids at one ormore of the following positions: position corresponding to any other oneof amino acids 1-181 from SEQ ID NO: 1 or before position 1 (i.e. at theN-terminus) or at position 182 (i.e., at the carboxyl terminus of theprotein) therein or the corresponding amino acids in SEQ ID NOs: 2-7 oranother modified FGF-21 polypeptide of the disclosure. In anotherembodiment, there is a non-naturally occurring amino acid at 91 and oneor more other non-naturally occurring amino acid at one or more of thefollowing positions: 131, 108, 77, 72, 87, 86, 126, 110, 83, 146, 135,96, and 36 (amino acid position corresponding to SEQ ID NO: 1 or thecorresponding amino acids of SEQ ID NOs: 2-7).

In another embodiment, there is a non-naturally occurring amino acid at131 and one other non-naturally occurring amino acid at one or more ofthe following positions: position corresponding to any other one ofamino acids 1-181 from SEQ ID NO: 1 or before position 1 (i.e. at theN-terminus) or at position 182 (i.e., at the carboxyl terminus of theprotein) therein or the corresponding amino acids in SEQ ID NOs: 2-7 oranother modified FGF-21 polypeptide of the disclosure. In anotherembodiment, there is a non-naturally occurring amino acid at 131 and oneother non-naturally occurring amino acid at one or more of the followingpositions: 131, 108, 77, 72, 87, 86, 126, 110, 83, 146, 135, 96, and 36(amino acid position corresponding to SEQ ID NO: 1 or the correspondingamino acids of SEQ ID NOs: 2-7).

In another embodiment, the amino acid sequence in the modified FGF-21polypeptide described herein may comprise a non-naturally encoded aminoacid at a position corresponding to residue 108 of SEQ ID NO: 1 and atleast two other non-naturally encoded amino acids at positionscorresponding to at least two of the following amino acids of SEQ ID NO:1: position corresponding to any other one of amino acids 1-181 from SEQID NO: 1 or before position 1 (i.e. at the N-terminus) or at position182 (i.e., at the carboxyl terminus of the protein) therein or thecorresponding amino acids in SEQ ID NOs: 2-7 or another modified FGF-21polypeptide of the disclosure. In another embodiment, the amino acidsequence in the modified FGF-21 polypeptide described herein maycomprise a non-naturally encoded amino acid at a position correspondingto amino acid 108 of SEQ ID NO: 1 and at least two other non-naturallyoccurring amino acids at positions corresponding to at least two of thefollowing amino acids of SEQ ID NO: 1: 10, 36, 52, 117, 126, 131, 135,146, 162, 87, 77, 83, 72, 69, 79, 91, 96, 108, or 110 of SEQ ID NO: 1.

In another embodiment, there is a non-naturally occurring amino acid at77 and one other non-naturally occurring amino acid at one or more ofthe following positions: position corresponding to any other one ofamino acids 1-181 from SEQ ID NO: 1 or before position 1 (i.e. at theN-terminus) or at position 182 (i.e., at the carboxyl terminus of theprotein) therein or the corresponding amino acids in SEQ ID NOs: 2-7 oranother modified FGF-21 polypeptide of the disclosure. In anotherembodiment, there is a non-naturally occurring amino acid at 77 and oneother non-naturally occurring amino acid at one or more of the followingpositions: 131, 108, 77, 72, 87, 86, 126, 110, 83, 146, 135, 96, and 36(SEQ ID NO: 1 or the corresponding amino acids of SEQ ID NOs: 2-7).

VII. Expression in Non-Eukaryotes and Eukaryotes I. Expression Systems,Culture, and Isolation

Unmodified or modified FGF-21 polypeptides may be expressed in anynumber of suitable expression systems including, for example, yeast,insect cells, mammalian cells, and bacteria. A description of exemplaryexpression systems is provided below.

Yeast: As used herein, the term “yeast” includes any of the variousyeasts capable of expressing a gene encoding a modified FGF-21polypeptide.

Of particular interest for use with the present disclosure are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula, Torulopsis, and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S. norbensis,S. oviformis, K. lactis, K fragilis, C. albicans, C. maltosa, and H.polymorpha. WO 2005/091944, which is incorporated by reference, hereindescribes the expression of FGF-21 in yeast.

Baculovirus-Infected Insect Cells: The term “insect host” or “insecthost cell” refers to a insect that can be, or has been, used as arecipient for recombinant vectors or other transfer DNA. The termincludes the progeny of the original insect host cell that has beentransfected. It is understood that the progeny of a single parental cellmay not necessarily be completely identical in morphology or in genomicor total DNA complement to the original parent, due to accidental ordeliberate mutation. Progeny of the parental cell that are sufficientlysimilar to the parent to be characterized by the relevant property, suchas the presence of a nucleotide sequence encoding a modified FGF-21polypeptide, are included in the progeny intended by this definition.

E. Coli, Pseudomonas Species, and Other Prokaryotes:

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding an unmodified or modified FGF-21polypeptide, are included in the progeny intended by this definition.

In selecting bacterial hosts for expression, suitable hosts may includethose shown to have, inter alia, good inclusion body formation capacity,low proteolytic activity, and overall robustness.Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). Other examples of suitable E. coli hosts include, but arenot limited to, strains of BL21, DH10B, or derivatives thereof. Inanother embodiment of the methods of the present disclosure, the E. colihost is a protease minus strain including, but not limited to, OMP- andLON-. The host cell strain may be a species of Pseudomonas, includingbut not limited to, Pseudomonas fluorescens, Pseudomonas aeruginosa, andPseudomonas putida. Pseudomonas fluorescens biovar 1, designated strainMB101, is known to be useful for recombinant production and is availablefor therapeutic protein production processes.

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of modified FGF-21 polypeptides.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where the modified FGF-21polypeptide accumulates intracellularly) or harvesting of culturesupernatant in either batch or continuous formats.

Modified FGF-21 polypeptides produced in bacterial host cells may bepoorly soluble or insoluble (in the form of inclusion bodies). In oneembodiment of the present disclosure, amino acid substitutions mayreadily be made in the modified FGF-21 polypeptide that are selected forthe purpose of increasing the solubility of the recombinantly producedprotein. The modified FGF-21 polypeptide may be solubilized, forexample, with urea or guanidine hydrochloride.

In the case of soluble modified FGF-21 protein, the FGF-21 may besecreted into the periplasmic space or into the culture medium. Forexample, modified FGF-21 is secreted into the periplasmic space ofW3110-B2 cells by using plasmids encoding constructs including eightdifferent leader sequences, including those listed in SEQ ID NOs: 39-44,and transforming these into W3110-B2 cells, the cells were then grown at37° C. until OD reached about 0.8, at which point the expression isinduced with 0.01% arabinose. Five hours later the periplasmic releasesamples can be prepped from the cultures. In addition, soluble modifiedFGF-21 may be present in the cytoplasm of the host cells. It may bedesired to concentrate soluble modified FGF-21 prior to performingpurification steps.

When modified FGF-21 polypeptide is produced as a fusion protein, thefusion sequence may be removed. Removal of a fusion sequence may beaccomplished by enzymatic or chemical cleavage. Enzymatic removal offusion sequences may be accomplished using methods known to those ofordinary skill in the art. The choice of enzyme for removal of thefusion sequence may be determined by the identity of the fusion, and thereaction conditions may be specified by the choice of enzyme as will beapparent to one of ordinary skill in the art. Chemical cleavage may beaccomplished using reagents known to those of ordinary skill in the art,including but not limited to, cyanogen bromide, TEV protease, and otherreagents. The cleaved modified FGF-21 polypeptide may be purified fromthe cleaved fusion sequence by methods known to those of ordinary skillin the art.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.The proteins can be refolded in a redox buffer containing, including butnot limited to, oxidized glutathione and L-arginine. Refolding reagentscan be flowed or otherwise moved into contact with the one or morepolypeptide or other expression product, or vice-versa.

In the case of prokaryotic production of modified FGF-21 polypeptide,the modified FGF-21 polypeptide thus produced may be misfolded and thuslacks or has reduced biological activity. The bioactivity of the proteinmay be restored by “refolding”. In general, misfolded unmodified ormodified FGF-21 polypeptide is refolded by solubilizing (where themodified FGF-21 polypeptide is also insoluble), unfolding and reducingthe polypeptide chain using, for example, one or more chaotropic agents(e.g. urea and/or guanidine) and a reducing agent capable of reducingdisulfide bonds (e.g. dithiothreitol, DTT or 2-mercaptoethanol, 2-ME).At a moderate concentration of chaotrope, an oxidizing agent is thenadded (e.g., oxygen, cystine or cystamine), which allows the reformationof disulfide bonds. Modified FGF-21 polypeptide may be refolded usingstandard methods known in the art, such as those described in U.S. Pat.Nos. 4,511,502, 4,511,503, and 4,512,922, which are incorporated byreference herein. The modified FGF-21 polypeptide may also be cofoldedwith other proteins to form heterodimers or heteromultimers.

After refolding, the modified FGF-21 may be further purified.Purification of modified FGF-21 may be accomplished using a variety oftechniques known to those of ordinary skill in the art, includinghydrophobic interaction chromatography, size exclusion chromatography,ion exchange chromatography, reverse-phase high performance liquidchromatography, affinity chromatography, and the like or any combinationthereof. Additional purification may also include a step of drying orprecipitation of the purified protein.

After purification, modified FGF-21 may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, diafiltration and dialysis. ModifiedFGF-21 that is provided as a single purified protein may be subject toaggregation and precipitation.

The purified modified FGF-21 may be at least 90% pure (as measured byreverse phase high performance liquid chromatography, RP-HPLC, or sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or atleast 95% pure, or at least 98% pure, or at least 99% or greater pure.Regardless of the exact numerical value of the purity of the modifiedFGF-21, the modified FGF-21 is sufficiently pure for use as apharmaceutical product or for further processing, such as conjugationwith a water soluble polymer such as PEG.

Certain modified FGF-21 molecules may be used as therapeutic agents inthe absence of other active ingredients or proteins (other thanexcipients, carriers, and stabilizers, serum albumin and the like), orthey may be complexed with another protein or a polymer.

In some embodiments of the present disclosure, the yield of modifiedFGF-21 after each purification step may be at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, at leastabout 99.9%, or at least about 99.99%, of the unmodified or modifiedFGF-21 in the starting material for each purification step.

VIII. Expression in Alternate Systems

Modified FGF-21 polypeptides of the present disclosure may be expressedusing a cell-free (e.g., in vitro) translational system. Translationsystems may be cellular or cell-free, and may be prokaryotic oreukaryotic. Cellular translation systems include, but are not limitedto, whole cell preparations such as permeabilized cells or cell cultureswherein a desired nucleic acid sequence can be transcribed to mRNA andthe mRNA translated. Cell-free translation systems are commerciallyavailable and many different types and systems are well-known. Examplesof cell-free systems include, but are not limited to, prokaryoticlysates such as Escherichia coli lysates, and eukaryotic lysates such aswheat germ extracts, insect cell lysates, rabbit reticulocyte lysates,rabbit oocyte lysates and human cell lysates. Membranous extracts, suchas the canine pancreatic extracts containing microsomal membranes, arealso available which are useful for translating secretory proteins.

IX. Macromolecular Polymers Coupled to Modified FGF-21 Polypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide; a water-soluble dendrimer; acyclodextrin; an inhibitory ribonucleic acid; a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety; a photoisomerizable moiety; biotin;a derivative of biotin; a biotin analogue; a moiety incorporating aheavy atom; a chemically cleavable group; a photocleavable group; anelongated side chain; a carbon-linked sugar; a redox-active agent; anamino thioacid; a toxic moiety; an isotopically labeled moiety; abiophysical probe; a phosphorescent group; a chemiluminescent group; anelectron dense group; a magnetic group; an intercalating group; achromophore; an energy transfer agent; a biologically active agent; adetectable label; a small molecule; a quantum dot; a nanotransmitter; aradionucleotide; a radiotransmitter; a neutron-capture agent; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to modified FGF-21 polypeptides of the present disclosure tomodulate biological properties of the modified FGF-21 polypeptide,and/or provide new biological properties to the modified FGF-21molecule. These macromolecular polymers can be linked to the modifiedFGF-21 polypeptide via a naturally encoded amino acid, via anon-naturally encoded amino acid, or any functional substituent of anatural or non-natural amino acid, or any substituent or functionalgroup added to a natural or non-natural amino acid. The molecular weightof the polymer may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof the polymer may be between about 100 Da and about 100,000 Da,including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da,50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and about 50,000Da. In some embodiments, the molecular weight of the polymer is betweenabout 100 Da and about 40,000 Da. In some embodiments, the molecularweight of the polymer is between about 1,000 Da and about 40,000 Da. Insome embodiments, the molecular weight of the polymer is between about5,000 Da and about 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 10,000 Da and about 40,000 Da.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.For therapeutic use of the end-product preparation, the polymer may bepharmaceutically acceptable.

Examples of polymers include but are not limited to polyalkyl ethers andalkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, especially polyoxyethylene glycol, the latter is also known aspolyethylene glycol or PEG); discrete PEG (dPEG); polyvinylpyrrolidones;polyvinylalkyl ethers; polyoxazolines, polyalkyl oxazolines andpolyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides, andpolyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide andderivatives thereof); polyhydroxyalkyl acrylates; polysialic acids andanalogs thereof; hydrophilic peptide sequences; polysaccharides andtheir derivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixturesthereof; and derivatives of the foregoing.

As used herein, and when contemplating PEG:modified FGF-21 polypeptideconjugates, the term “therapeutically effective amount” refers to anamount which gives the desired benefit to a patient. The amount may varyfrom one individual to another and may depend upon a number of factors,including the overall physical condition of the patient and theunderlying cause of the condition to be treated. The amount of modifiedFGF-21 polypeptide used for therapy gives an acceptable rate of changeand maintains desired response at a beneficial level.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this disclosure.

The term “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented as linked to the modified FGF-21 polypeptideby the formula:

XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y

where n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalfunctional group.

In some cases, a PEG used in the polypeptides of the disclosureterminates on one end with hydroxy or methoxy, i.e., X is H or CH₃(“methoxy PEG”). Alternatively, the PEG can terminate with a reactivegroup, thereby forming a bifunctional polymer. Typical reactive groupscan include those reactive groups that are commonly used to react withthe functional groups found in the 20 common amino acids (including butnot limited to, maleimide groups, activated carbonates (including butnot limited to, p-nitrophenyl ester), activated esters (including butnot limited to, N-hydroxysuccinimide, p-nitrophenyl ester) andaldehydes) as well as functional groups that are inert to the 20 commonamino acids but that react specifically with complementary functionalgroups present in non-naturally encoded amino acids (including but notlimited to, azide groups, alkyne groups). It is noted that the other endof the PEG, which is shown in the above formula by Y, may attach eitherdirectly or indirectly to a modified FGF-21 polypeptide via anaturally-occurring or non-naturally encoded amino acid. For instance, Ymay be an amide, carbamate or urea linkage to an amine group (includingbut not limited to, the epsilon amine of lysine or the N-terminus) ofthe polypeptide. Alternatively, Y may be a maleimide linkage to a thiolgroup (including but not limited to, the thiol group of cysteine).Alternatively, Y may be a linkage to a residue not commonly accessiblevia the 20 common amino acids. For example, an azide group on the PEGcan be reacted with an alkyne group on the modified FGF-21 polypeptideto form a Huisgen [3+2] cycloaddition product. Alternatively, an alkynegroup on the PEG can be reacted with an azide group present in anon-naturally encoded amino acid to form a similar product. In someembodiments, a strong nucleophile (including but not limited to,hydrazine, hydrazide, hydroxylamine, semicarbazide) can be reacted withan aldehyde or ketone group present in a non-naturally encoded aminoacid to form a hydrazone, oxime or semicarbazone, as applicable, whichin some cases can be further reduced by treatment with an appropriatereducing agent. Alternatively, the strong nucleophile can beincorporated into the modified FGF-21 polypeptide via a non-naturallyencoded amino acid and used to react preferentially with a ketone oraldehyde group present in the water soluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). The molecular weight of PEG may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. PEG may be between about 100 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500Da, 400 Da, 300 Da, 200 Da, and 100 Da. Branched chain PEGs, includingbut not limited to, PEG molecules with each chain having a MW rangingfrom 1-100 kDa (including but not limited to, 1-50 kDa or 5-20 kDa) canalso be used. The molecular weight of each chain of the branched chainPEG may be, including but not limited to, between about 1,000 Da andabout 100,000 Da or more. The molecular weight of each chain of thebranched chain PEG may be between about 1,000 Da and about 100,000 Da,including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da,50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. Generally, at least oneterminus of the PEG molecule is available for reaction with thenon-naturally-encoded amino acid. For example, PEG derivatives bearingalkyne and azide moieties for reaction with amino acid side chains canbe used to attach PEG to non-naturally encoded amino acids as describedherein. If the non-naturally encoded amino acid comprises an azide, thenthe PEG may typically contain either an alkyne moiety to effectformation of the [3+2] cycloaddition product or an activated PEG species(i.e., ester, carbonate) containing a phosphine group to effectformation of the amide linkage. Alternatively, if the non-naturallyencoded amino acid comprises an alkyne, then the PEG may typicallycontain an azide moiety to effect formation of the [3+2] Huisgencycloaddition product. If the non-naturally encoded amino acid comprisesa carbonyl group, the PEG may typically comprise a potent nucleophile(including but not limited to, a hydrazide, hydrazine, hydroxylamine, orsemicarbazide functionality) in order to effect formation ofcorresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the modified FGF-21 polypeptide with a PEGderivative contains a chemical functionality that is reactive with thechemical functionality present on the side chain of the non-naturallyencoded amino acid.

The disclosure provides in some embodiments azide- andacetylene-containing polymer derivatives comprising a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da. The polymer backbone of the water-soluble polymer canbe poly(ethylene glycol). However, it should be understood that a widevariety of water soluble polymers including but not limited topoly(ethylene)glycol and other related polymers, including poly(dextran)and poly(propylene glycol), are also suitable for use in the presentlydisclosed polypeptides and that the use of the term PEG or poly(ethyleneglycol) is intended to encompass and include all such molecules.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. Branched PEG can also be inthe form of a forked PEG represented by PEG(-YCHZ₂)_(n), where Y is alinking group and Z is an activated terminal group linked to CH by achain of atoms of defined length. Yet another branched form, the pendantPEG, has reactive groups, such as carboxyl, along the PEG backbonerather than at the end of PEG chains.

Many other polymers are also suitable for use in the polypeptides of thepresent disclosure. Examples of suitable polymers include, but are notlimited to, other poly(alkylene glycols), such as poly(propylene glycol)(“PPG”), copolymers thereof (including but not limited to copolymers ofethylene glycol and propylene glycol), terpolymers thereof, mixturesthereof, and the like. Although the molecular weight of each chain ofthe polymer backbone can vary, it is typically in the range of fromabout 800 Da to about 100,000 Da, often from about 6,000 Da to about80,000 Da. The molecular weight of each chain of the polymer backbonemay be between about 100 Da and about 100,000 Da, including but notlimited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da.

Water soluble polymers can be linked to the modified FGF-21 polypeptidesof the disclosure. The water soluble polymers may be linked via anon-naturally encoded amino acid incorporated in the modified FGF-21polypeptide or any functional group or substituent of a non-naturallyencoded or naturally encoded amino acid, or any functional group orsubstituent added to a non-naturally encoded or naturally encoded aminoacid. Alternatively, the water soluble polymers are linked to a modifiedFGF-21 polypeptide comprising a non-naturally encoded amino acid via anaturally-occurring amino acid (including but not limited to, cysteine,lysine or the amine group of the N-terminal residue). In some cases, themodified FGF-21 polypeptides of the disclosure comprise 1, 2, 3, 4, 5,6, 7, 8, 9, 10 non-natural amino acids, wherein one or morenon-naturally-encoded amino acid(s) are linked to water solublepolymer(s) (including but not limited to, PEG and/or oligosaccharides).In some cases, the modified FGF-21 polypeptides of the disclosurefurther comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morenaturally-encoded amino acid(s) linked to water soluble polymers. Insome cases, the modified FGF-21 polypeptides of the disclosure compriseone or more non-naturally encoded amino acid(s) linked to water solublepolymers and one or more naturally-occurring amino acids linked to watersoluble polymers. In some embodiments, the water soluble polymers usedin the present disclosure enhance the serum half-life of the modifiedFGF-21 polypeptide relative to a comparator compound such as the FGF-21polypeptide of SEQ ID NO:1, SEQ ID NO:201, the same FGF-21 polypeptidewithout said water soluble polymer, or another comparator compounddescribed elsewhere herein.

The number of water soluble polymers linked to a modified FGF-21polypeptide (i.e., the extent of PEGylation or glycosylation) of thepresent disclosure can be adjusted to provide an altered (including butnot limited to, increased or decreased) pharmacologic, pharmacokineticor pharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of modified FGF-21 is increased at leastabout 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold,30-fold, 35-fold, 40-fold, 50-fold, or at least about 100-fold over anunmodified polypeptide.

PEG Derivatives Containing a Strong Nucleophilic Group (i.e., Hydrazide,Hydrazine, Hydroxylamine or Semicarbazide)

In one embodiment of the present disclosure, a modified FGF-21polypeptide comprising a carbonyl-containing non-naturally encoded aminoacid may be modified with a PEG derivative that contains a terminalhydrazine, hydroxylamine, hydrazide or semicarbazide moiety that islinked directly to the PEG backbone.

The degree and sites at which the water soluble polymer(s) are linked tothe modified FGF-21 polypeptide can modulate the binding of the modifiedFGF-21 polypeptide to the FGF-21 polypeptide receptor. In someembodiments, the linkages may be arranged such that the modified FGF-21polypeptide binds the FGF-21 polypeptide receptor with a K_(d) of about400 nM or lower, with a K_(d) of 150 nM or lower, and in some cases witha K_(d) of 100 nM or lower, as measured by an equilibrium binding assay,such as that described in Spencer et al., J. Biol. Chem., 263:7862-7867(1988) for modified FGF-21.

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.PEGylation (i.e., addition of any water soluble polymer) of modifiedFGF-21 polypeptides containing a non-naturally encoded amino acid, suchas p-azido-L-phenylalanine, may be carried out by any suitable method,such as those described in U.S. Pat. No. 8,012,931, which isincorporated by reference herein. A water soluble polymer linked to anamino acid of a modified FGF-21 polypeptide of the disclosure can befurther derivatized or substituted without limitation.

In another embodiment of the disclosure, a modified FGF-21 polypeptidemay be modified with a PEG derivative that contains an azide moiety thatmay react with an alkyne moiety present on the side chain of thenon-naturally encoded amino acid. In general, the PEG derivatives mayhave an average molecular weight ranging from 1-100 kDa and, in someembodiments, from 10-40 kDa.

In some embodiments, the azide-terminal PEG derivative may have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment, the azide-terminal PEG derivative may have thestructure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40kDa).

In another embodiment of the disclosure, a modified FGF-21 polypeptidecomprising a alkyne-containing amino acid may be further modified with abranched PEG derivative that contains a terminal azide moiety, with eachchain of the branched PEG having a MW ranging from 10-40 kDa and may befrom 5-20 kDa. For instance, in some embodiments, the azide-terminal PEGderivative may have the following structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)N₃

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), in each case that can be present or absent.

In another embodiment of the disclosure, a modified FGF-21 polypeptidemay be modified with a PEG derivative that contains an alkyne moietythat may react with an azide moiety present on the side chain of thenon-naturally encoded amino acid.

In another embodiment of the disclosure, a modified FGF-21 polypeptidemay be modified with a PEG derivative that contains an activatedfunctional group (including but not limited to, ester, carbonate)further comprising an aryl phosphine group that may react with an azidemoiety present on the side chain of the non-naturally encoded aminoacid.

A modified FGF-21 polypeptide may be conjugated to ahydrazide-containing PEG by the following exemplary methods. A modifiedFGF-21 polypeptide incorporating a carbonyl-containing amino acid (suchas pAcF) may be prepared according to the procedure described above.Once modified, a hydrazide-containing PEG having the following structuremay be conjugated to the modified FGF-21 polypeptide:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—X—NH—NH₂

where, for example, R=methyl, n=2 and N=10,000 MW and X is a carbonyl(C═O) group. The purified modified FGF-21 containingp-acetylphenylalanine is dissolved at between 0.1-10 mg/mL in 25 mM MES(Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, Mo.) pH 4.5, is reacted with a 1 to 100-fold excess ofhydrazide-containing PEG, and the corresponding hydrazone is reduced insitu by addition of stock 1M NaCNBH₃ (Sigma Chemical, St. Louis, Mo.),dissolved in H₂O, to a final concentration of 10-50 mM. Reactions arecarried out in the dark at 4° C. to RT for 18-24 hours. Reactions arestopped by addition of 1 M Tris (Sigma Chemical, St. Louis, Mo.) atabout pH 7.6 to a final Tris concentration of 50 mM or diluted intoappropriate buffer for immediate purification.

Introduction Ofan Alkyne-Containing Amino Acid into a Modified FGF-21Polypeptide and Derivatization with Mpeg-Azide

Modified FGF-21 polypeptides containing an alkyne-containing amino acidmay be produced and may be derivatized with mPEG-azide by the followingexemplary methods. A selected position may be substituted with thefollowing non-naturally encoded amino acid:

The sequences utilized for site-specific incorporation ofp-propargyl-tyrosine into modified FGF-21 may be SEQ ID NO: 1 (FGF-21),SEQ ID NO: 16 or 17 (muttRNA, M. jannaschii mtRNA_(CUA) ^(Tyr)), 22, 23or 24 described above. The modified FGF-21 polypeptide containing thepropargyl tyrosine may be expressed in E. coli and purified using theconditions described above for purification of a modified FGF-21polypeptide containing a carbonyl-containing amino acid.

The purified modified FGF-21 containing propargyl-tyrosine dissolved atbetween 0.1-10 mg/mL in PB buffer (100 mM sodium phosphate, 0.15 M NaCl,pH=8) and a 10 to 1000-fold excess of an azide-containing PEG is addedto the reaction mixture. A catalytic amount of CuSO₄ and Cu wire arethen added to the reaction mixture. After the mixture is incubated(including but not limited to, about 4 hours at room temperature or 37°C., or overnight at 4° C.), H₂O is added and the mixture is filteredthrough a dialysis membrane. The sample can be analyzed to confirmyield.

X. Fusion Proteins Containing Modified FGF-21 Polypeptides

The disclosure also provides modified FGF-21 polypeptides, or afragments thereof, comprising the modified FGF-21 polypeptide sequenceand a fusion partner. The fusion partner may confer a functionalproperty, including but not limited to, half-life extension,facilitating protein purification and/or manufacturing, enhancedbiophysical properties such as increase solubility or stability, andreduced immunogenicity or toxicity, or any other purpose. For example,the fusion protein may exhibit extended in vivo half-life, therebyfacilitating a less frequent dosing (such as dosing twice per week, onceper week, or once every other week, etc.) in a therapeutic regimen.Exemplary fusion proteins comprise a modified FGF-21 fused to a fusionpartner such as an albumin (e.g., human serum albumin), PK extending(PKE) adnectin, XTEN, Fc domain, or a fragment of any of the foregoing,or a combination of any of the foregoing. A fusion protein can beproduced by expressing a nucleic acid which encodes the modified FGF-21polypeptide sequence and a fusion partner sequence in the same readingframe, optionally separated by a sequence encoding a connecting peptide.The fusion protein may comprise the modified FGF-21 polypeptide andfusion partner in any order, e.g., one or more fusion partners linked tothe N-terminus and/or C-terminus of the modified FGF-21 polypeptidesequence, or one or more fusion partners linked to both the N-terminusand C-terminus.

The fusion may be formed by attaching a fusion partner to either end(i.e., either the N- or C-terminus) of a modified FGF-21 polypeptide,i.e., fusion partner-modified FGF-21 or modified FGF-21-fusion partnerarrangements. Additionally, the modified FGF-21 polypeptide may be fusedto one or more fusion partners at both ends, optionally with aconnecting peptide at either end or both ends. In certain embodiments,the fusion partner and modified FGF-21 are fused via a connectingpeptide. Exemplary connecting peptides may comprise or consist of asequence selected from SEQ ID NOs:74-100, 301, and 350-383, or acombination of the foregoing (e.g., two, three, or more of the foregoingsequences). Exemplary connecting peptides can have lengths of between 0(i.e., no connecting peptide present) and 100 or more amino acids, suchas between at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and up to 60, 50,40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, or 11 amino acids. Exemplary non-limiting lengths of aconnecting peptide include between 1 and 100 amino acids, 1 and 40 aminoacids, between 1 and 20 amino acids, between 1 and 10 amino acids, orbetween 3 and 5 amino acids in length.

A modified FGF-21 polypeptide, or a fragment thereof, can be produced asa fusion protein comprising human serum albumin (HSA) or a portionthereof. Such fusion constructs may be suitable for enhancing expressionof the modified FGF-21, or fragment thereof, in a eukaryotic host cell,such as CHO, or in a bacterium such as E. coli. Exemplary HSA portionsinclude the N-terminal polypeptide (amino acids 1-369, 1-419, andintermediate lengths starting with amino acid 1), as disclosed in U.S.Pat. No. 5,766,883, and PCT publication WO 97/24445, which isincorporated by reference herein. In some embodiments, the fusionprotein may comprise a HSA protein with a modified FGF-21, or fragmentsthereof, attached to each of the C-terminal and N-terminal ends of theHSA. Exemplary HSA constructs are disclosed in U.S. Pat. No. 5,876,969,which is incorporated by reference herein. Exemplary methods ofmammalian cell expression of FGF-21 are described in WO 2005/091944which is incorporated by reference herein.

The modified FGF-21 polypeptide may be fused an XTEN molecule. XTENmolecules are also referred to as unstructured recombinant polymers,unstructured recombinant polypeptides (URPs), and are generallydescribed in Schellenberger et al., Nat Biotechnol., 2009 December;27(12):1186-90, U.S. Pub. No. 2012/0220011, U.S. Pat. No. 7,846,445, andWO/2012/162542, each of which is hereby incorporated by reference in itsentirety. The half-life of the modified FGF-21 polypeptide may be variedby varying the constitution of the XTEN molecule, e.g., by varying itssize. For example, an XTEN molecule may be selected in order to achievea desired half-life, such as in the range of 1 to 50 hours, such as atleast 1, 2, 5, 10, 12, 15, 20, or 25 hours, or longer.

Exemplary XTEN molecules include a URP comprising at least 40 contiguousamino acids, wherein: (a) the URP comprises at least three differenttypes of amino acids selected from the group consisting of glycine (G),aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) andproline (P) residues, wherein the sum of said group of amino acidscontained in the URP constitutes more than about 80% of the total aminoacids of the URP, and wherein said URP comprises more than one prolineresidue, and wherein said URP possesses reduced sensitivity toproteolytic degradation relative to a corresponding URP lacking saidmore than one proline residue; (b) at least 50% of the amino acids ofsaid URP are devoid of secondary structure as determined by Chou-Fasmanalgorithm; and (c) the Tepitope score of said URP is less than −5.Additional exemplary XTEN molecules comprise an unstructured recombinantpolymer (URP) comprising at least about 40 contiguous amino acids, andwherein (a) the sum of glycine (G), aspartate (D), alanine (A), serine(S), threonine (T), glutamate (E) and proline (P) residues contained inthe URP, constitutes at least 80% of the total amino acids of the URP,and the remainder, when present, consists of arginine or lysine, and theremainder does not contain methionine, cysteine, asparagine, andglutamine, wherein said URP comprises at least three different types ofamino acids selected from glycine (G), aspartate (D), alanine (A),serine (S), threonine (T), glutamate (E) and proline (P); (b) at least50% of the at least 40 contiguous amino acids in said URP are devoid ofsecondary structure as determined by Chou-Fasman algorithm; and (c)wherein the URP has a Tepitope score less than −4.

Additional exemplary XTEN molecules include rPEG molecules. In someembodiments, the rPEG molecule may not include a hydrophobic residue(e.g., F, I, L, M, V, W or Y), a side chain amide-containing residue(e.g., N or Q) or a positively charged side chain residue (e.g., H, K orR). In some embodiments, the enhancing moiety may include A, E, G, P, Sor T. In some embodiments, the rPEG may include glycine at 10-20%,20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or evenglycine at 100%.

Said XTEN molecule may be further linked to a polyethylene glycol.

The term “PAS” and/or “PASylation” refers to the genetic fusion of abiopharmaceutical protein of interest such as the modified FGF-21polypeptide with a conformationally disordered polypeptide sequencecomposed of the amino acids Pro, Ala and Ser (hence the term “PASpolypeptide” and “PASylation”). PASylation may increase the serum-halflife, increased solubility, stability, and/or resistance to protease,and/or decreased immunogenicity of the protein of interest, e.g. thefusion protein (for reference, see WO2008155134 A1 and US20140073563,which are incorporated herein by reference). The PAS sequences may beattached via gene fusion to either the N-terminus, the C-terminus or toboth termini of the amino acid sequence of the modified FGF-21polypeptide. In some embodiments, a PAS polypeptide may comprise atleast two domains comprises an amino acid sequence consisting of atleast about 100, 150, 200, 250, 300, 350,400, 450, 500, 600, 700, 800,900, 1000, or more amino acid residues forming random coil conformationand wherein said second domain comprising alanine, serine and prolineresidues, whereby said random coil conformation mediates an increased invivo and/or in vitro stability of the biopharmaceutical protein to whichthe PAS polypeptide is fused. In some embodiments, the second domaincomprising alanine, serine and proline residues may be selected fromASPAAPAPASPAAPAPSAPA (SEQ ID NO: 310); AAPASPAPAAPSAPAPAAPS (SEQ ID NO:311); APSSPSPSAPSSPSPASPSS (SEQ ID NO: 312), SAPSSPSPSAPSSPSPASPS (SEQID NO: 313), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 314),AASPAAPSAPPAAASPAAPSAPPA 1.1) NO: 315) and ASAAAPAAASAAASAPSAAA (SEQ1.1) NO: 316). The PAS polypeptide may contain one or more site(s) forcovalent modification.

In exemplary embodiments the modified FGF-21 is fused to an adnectin,e.g. an albumin-binding or PKE adnectin. Exemplary adnectins aredisclosed in U.S. Pub. No. 2011/0305663, which is hereby incorporated byreference in its entirety. Said adnectin may be based on a tenthfibronectin type III domain and may bind to serum albumin. Said adnectinmay comprise one or more of a BC loop comprising the amino acid sequenceset forth in SEQ ID NO: 45, a DE loop comprising the amino acid sequenceset forth in SEQ ID NO: 46, and an FG loop comprising the amino acidsequence set forth in SEQ ID NO: 47, or comprises a polypeptide selectedfrom SEQ ID NO: 48, 49, 50, 51, and 52-72, or comprises a polypeptide atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90% or at least 95% identical to SEQ ID NO: 48, 49, 50, 51, or52-72, which respectively correspond to SEQ ID NOS 5, 6, 7, 8, 12, 16,20, and 24-44 of U.S. Pub. No. 2011/0305663.

In some embodiments, the modified FGF-21 polypeptide may be fused to animmunoglobulin Fc domain (“Fc domain”), or a fragment or variantthereof, such as a functional Fc region. A functional Fc region binds toFcRn, but does not possess effector function. The ability of the Fcregion or fragment thereof to bind to FcRn can be determined by standardbinding assays known in the art. Exemplary “effector functions” includeC1q binding; complement dependent cytotoxicity (CDC); Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down regulation of cell surface receptors (e.g., B cellreceptor; BCR), etc. Such effector functions can be assessed usingvarious assays known in the art for evaluating such antibody effectorfunctions.

In an exemplary embodiment, the Fc domain is derived from an IgG1subclass, however, other subclasses (e.g., IgG2, IgG3, and IgG4) mayalso be used. Shown below is an exemplary sequence of a human IgG1immunoglobulin Fc domain:

(SEQ ID NO: 302) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some embodiments, the Fc region used in the fusion protein maycomprise the hinge region of an Fc molecule. An exemplary hinge regioncomprises the core hinge residues spanning positions 1-16 (i.e.,DKTHTCPPCPAPELLG (SEQ ID NO:303)) of the exemplary human IgG1immunoglobulin Fc domain sequence provided above. In certainembodiments, the fusion protein may adopt a multimeric structure (e.g.,dimer) owing, in part, to the cysteine residues at positions 6 and 9within the hinge region of the exemplary human IgG1 immunoglobulin Fcdomain sequence provided above. In other embodiments, the hinge regionas used herein, may further include residues derived from the CH1 andCH2 regions that flank the core hinge sequence of the exemplary humanIgG1 immunoglobulin Fc domain sequence provided above. In yet otherembodiments, the hinge sequence may comprise or consist ofGSTHTCPPCPAPELLG (SEQ ID NO:304).

In some embodiments, the hinge sequence may include one or moresubstitutions that confer desirable pharmacokinetic, biophysical, and/orbiological properties. Some exemplary hinge sequences includeEPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO:305), EPKSSDKTHTCPPCPAPELLGGSS (SEQID NO:306), EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO:307),DKTHTCPPCPAPELLGGPS (SEQ ID NO:308), and DKTHTCPPCPAPELLGGSS (SEQ IDNO:309). In one embodiment, the residue P at position 18 of theexemplary human IgG1 immunoglobulin Fc domain sequence provided abovemay be replaced with S to ablate Fc effector function; this replacementis exemplified in hinges having the sequences EPKSSDKTHTCPPCPAPELLGGSS(SEQ ID NO:306), EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO:307), andDKTHTCPPCPAPELLGGSS (SEQ ID NO:309). In another embodiment, the residuesDK at positions 1-2 of the exemplary human IgG1 immunoglobulin Fc domainsequence provided above may be replaced with GS to remove a potentialclip site; this replacement is exemplified in the sequenceEPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO:307). In another embodiment, the Cat the position 103 of the heavy chain constant region of human IgG1(i.e., domains CH₁-CH₃), may be replaced with S to prevent impropercysteine bond formation in the absence of a light chain; thisreplacement is exemplified in the sequences EPKSSDKTHTCPPCPAPELLGGPS(SEQ ID NO:305), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO:306), andEPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO:307).

Additional exemplary Fc sequences include the following:

hIgG1a_191 [A subtype] (SEQ ID NO: 323)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1a_189 [hIgG1a_191 sans “GK”on C term; A subtype] (SEQ ID NO: 324)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP hIgG1a_191b [A/F subtype](SEQ ID NO: 325) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKhIgG1f_1.1_191 [Contains 5 point mutations toalter ADCC function, F subtype] (SEQ ID NO: 326)DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKhIgG1f_1.1_186 [Contains 5 point mutations to alter ADCC function and C225S (Edlemen numbering); F subtype](SEQ ID NO: 327) EPKSS DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1a_(N297G)_191 [A subtype](SEQ ID NO: 328) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1a_190 [hIgG1a_190 sans “K”on C term; A subtype] (SEQ ID NO: 329)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG hIgG1a_(N297Q)_191 [A subtype](SEQ ID NO: 330) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1a_(N297S)_191 [A subtype](SEQ ID NO: 331) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1a_(N297A)_191 [A subtype](SEQ ID NO: 332) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1a_(N297H)_191 [A subtype](SEQ ID NO: 333) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYHSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG4 (SEQ ID NO: 334) DKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK hIgG4_(S241P) (SEQ ID NO: 335)DKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

In some embodiments, the Fc domain comprises an amino acid sequenceselected from SEQ ID NOs: 302 and 323-335. It should be understood thatthe C-terminal lysine of an Fc domain is an optional component of afusion protein comprising an Fc domain. In some embodiments, the Fcdomain comprises an amino acid sequence selected from SEQ ID NOs: 302and 323-335, except that the C-terminal lysine thereof is omitted. Insome embodiments, the Fc domain comprises the amino acid sequence of SEQID NO: 302. In some embodiments, the Fc domain comprises the amino acidsequence of SEQ ID NOs: 302 except the C-terminal lysine thereof isomitted.

In some embodiments, the modified FGF-21 polypeptide comprising analbumin binding sequence is made. Exemplary albumin binding sequencesinclude, but are not limited to, the albumin binding domain fromstreptococcal protein G (see. e.g., Makrides et al., J. PharmacoL Exp.Ther. 277:534-542 (1996) and Sjolander et al., J, Immunol. Methods201:115-123 (1997)), or albumin-binding peptides such as those describedin, e.g., Dennis, et al., J. Biol. Chem. 277:35035-35043 (2002).

In some embodiments, the modified FGF-21 polypeptides of the presentdisclosure are acylated with fatty acids. In some cases, the fatty acidspromote binding to serum albumin. See, e.g., Kurtzhals, et al., Biochem.J. 312:725-731 (1995).

In some embodiments, the modified FGF-21 polypeptides of the presentdisclosure are fused directly with serum albumin (including but notlimited to, human serum albumin) Those of skill in the art willrecognize that a wide variety of other molecules can also be linked tomodified FGF-21 in the present disclosure to modulate binding to serumalbumin or other serum components.

XI. Glycosylation of Modified and Unmodified FGF-21 Polypeptides

The present disclosure includes modified FGF-21 polypeptides comprisingone or more non-naturally encoded amino acids bearing saccharideresidues. The saccharide residues may be either natural (including butnot limited to, N-acetylglucosamine) or non-natural (including but notlimited to, 3-fluorogalactose). The saccharides may be linked to thenon-naturally encoded amino acids either by an N- or O-linked glycosidiclinkage (including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C- or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to modified FGF-21 polypeptides either in vivo or in vitro. Insome embodiments of the present disclosure, a modified FGF-21polypeptide comprising a carbonyl-containing non-naturally encoded aminoacid may be modified with a saccharide derivatized with an aminooxygroup to generate the corresponding glycosylated polypeptide linked viaan oxime linkage. Once attached to the non-naturally encoded amino acid,the saccharide may be further elaborated by treatment withglycosyltransferases and other enzymes to generate an oligosaccharidebound to the modified FGF-21 polypeptide. See, e.g., H. Liu, et al. J.Am. Chem. Soc. 125: 1702-1703 (2003).

In some embodiments of the present disclosure, a modified FGF-21polypeptide comprising a carbonyl-containing non-naturally encoded aminoacid may be modified directly with a glycan with defined structureprepared as an aminooxy derivative. Other functionalities, includingazide, alkyne, hydrazide, hydrazine, and semicarbazide, can be used tolink the saccharide to the non-naturally encoded amino acid.

In some embodiments of the present disclosure, a modified FGF-21polypeptide comprising an azide or alkynyl-containing non-naturallyencoded amino acid can then be modified by, including but not limitedto, a Huisgen [3+2] cycloaddition reaction with, including but notlimited to, alkynyl or azide derivatives, respectively.

XII. FGF-21 Dimers and Multimers

The present disclosure also provides for FGF-21 and modified FGF-21combinations such as homodimers, heterodimers, homomultimers, orheteromultimers (i.e., timers, tetramers, etc.) where modified FGF-21containing one or more non-naturally encoded amino acids is bound toanother FGF-21 or modified FGF-21 or a variant thereof or any otherpolypeptide that is not FGF-21 or modified FGF-21 or a variant thereof,either directly to the polypeptide backbone or via a linker. Due to itsincreased molecular weight compared to monomers, the FGF-21 dimer ormultimer conjugates may exhibit new or desirable properties, includingbut not limited to different pharmacological, pharmacokinetic,pharmacodynamic, modulated therapeutic half-life, or modulated plasmahalf-life relative to the monomeric FGF-21. In some embodiments,modified FGF-21 dimers of the present disclosure may modulate signaltransduction of the FGF-21 receptor. In other embodiments, the modifiedFGF-21 dimers or multimers of the present disclosure may act as a FGF-21receptor antagonist, agonist, or modulator.

XIII. Measurement of FGF-21 Polypeptide Activity and Affinity of FGF-21Polypeptide for the FGF-21 Polypeptide Receptor

FGF-21 has been shown to stimulate glucose uptake and enhance insulinsensitivity in 3T3-L1 adipocytes, an in vitro model utilized for thestudy of adipose tissue metabolism as shown in Example 3 of U.S. PatentPublication No. 20040259780 which is incorporated by reference in itsentirety. A characteristic of Type 2 diabetes is the deficiency ofglucose uptake in various tissue types including adipose tissue. Thus,modified FGF-21 may be useful for treating Type 2 diabetes by loweringblood glucose levels. Moreover, modified FGF-21 may be useful fortreating obesity by increasing energy expenditure by faster and moreefficient glucose utilization. Additionally, FGF-21 has been shown tostimulate glucose uptake in 3T3-L1 adipocytes in an insulin independentmanner, indicating that it is useful for treating Type 1 diabetes aswell. See U.S. Patent Publication No. 20040259780. FGF-21 is shown tostimulate glucose uptake in 3T3-L1 adipocytes in a concentrationdependent manner at a sub-optimal concentration of insulin (5 nM) and inthe absence of insulin in U.S. Patent Publication No. 20040259780.Additionally, FGF-21 induces glucose uptake in an ex vivo tissue model,described in U.S. Patent Publication No. 20040259780.

The modified FGF-21 polypeptides may be subject to assays for biologicalactivity. In general, the test for biological activity should provideanalysis for the desired result, such as increase or decrease inbiological activity, different biological activity, receptor or bindingpartner affinity analysis, conformational or structural changes of themodified FGF-21 itself or its receptor, or serum half-life analysis, ascompared to unmodified FGF-21 or another comparator compound asdescribed elsewhere herein.

Exemplary methods for differentiation of 3T3-L1 to adipocytes andglucose uptake assay are described in U.S. Pat. No. 8,012,931, which isincorporated herein by reference in its entirety.

XIV. Measurement of Potency, Functional In Vivo Half-Life, andPharmacokinetic Parameters

An aspect of the present disclosure is the prolonged biologicalhalf-life that can be obtained by construction of the modified FGF-21polypeptide, which may be conjugated to a water soluble polymer moiety,or fused to a fusion partner. The rapid post administration decrease ofFGF-21 polypeptide serum concentrations has made it therapeuticallysignificant to evaluate biological responses to treatment with themodified FGF-21 polypeptide. The modified FGF-21 polypeptide of thepresent disclosure may have prolonged serum half-lives also afteradministration via, e.g. subcutaneous or i.v. administration, making itpossible to measure by, e.g. ELISA method or by a primary screeningassay. Measurement of in vivo biological half-life is carried out asdescribed herein.

The potency and functional in vivo half-life of the modified FGF-21polypeptide comprising an internal deletion and/or non-naturally encodedamino acid may be determined according to protocols known to those ofordinary skill in the art and as described herein.

In an exemplary assay, pharmacokinetic parameters for a unmodified ormodified FGF-21 polypeptide described herein can be evaluated in normalSprague-Dawley male rats (N=5 animals per treatment group). Animals mayreceive either a single dose of 25 ug/rat iv or 50 ug/rat sc, andapproximately 5-7 blood samples may be taken according to a pre-definedtime course, generally covering about 6 hours for a modified FGF-21polypeptide comprising a non-naturally encoded amino acid not conjugatedto a water soluble polymer and about 4 days for a modified FGF-21polypeptide comprising a non-naturally encoded amino acid and conjugatedto a water soluble polymer. Pharmacokinetic data for a modified FGF-21can be compared to a comparator compound such as a wild-type FGF-21polypeptide of SEQ ID NO:1, the modified FGF-21 polypeptide of SEQ IDNO:201, the same modified FGF-21 polypeptide lacking an internaldeletion, or another comparator compound described herein.

Pharmacokinetic parameters can also be evaluated in a primate, e.g.,cynomolgus monkeys. Typically, a single injection is administered eithersubcutaneously or intravenously, and serum FGF-21 levels are monitoredover time.

Polypeptides of the present disclosure may be used to treat mammalssuffering from non-insulin dependent Diabetes Mellitus (NIDDM: Type 2),insulin dependent diabetes (Type 1), as well as obesity, inadequateglucose clearance, hyperglycemia, hyperinsulinemia, and the like. FGF-21is effective in animal models of diabetes and obesity, as shown in U.S.Patent Publication No. 20040259780, which is incorporated by referenceherein in its entirety. As metabolic profiles differ among variousanimal models of obesity and diabetes, analysis of multiple models havebeen undertaken to separate the effects of hyperinsulinemia,hyperglycemia and obesity. The diabetes (db/db) and obese (ob/ob) miceare characterized by massive obesity, hyperphagia, variablehyperglycemia, insulin resistance, hyperinsulinemia and impairedthermogenesis (Coleman, Diabetes 31:1, 1982; E. Shafrir, in DiabetesMellitus; H. Rifkin and D. Porte, Jr. Eds. (Elsevier Science PublishingCo., Inc., New York, ed. 4, 1990), pp. 299-340). However, diabetes ismuch more severe in the db/db model (Coleman, Diabetes 31:1, 1982; E.Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.(Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990), pp.299-340). Zucker (fa/fa) rats are severely obese, hyperinsulinemic, andinsulin resistant (Coleman, Diabetes 31:1, 1982; E. Shafrir, in DiabetesMellitus; H. Rifkin and D. Porte, Jr. Eds. (Elsevier Science PublishingCo., Inc., New York, ed. 4, 1990), pp. 299-340), and the fa/fa mutationmay be the rat equivalent of the murine db mutation (Friedman et al.,Cell 69:217-220, 1992; Truett et al., Proc. Natl. Acad. Sci. USA88:7806, 1991). Tubby (tub/tub) mice are characterized by obesity,moderate insulin resistance and hyperinsulinemia without significanthyperglycemia (Coleman et al., J. Heredity 81:424, 1990).

The monosodium glutamate (MSG) model for chemically-induced obesity(Olney, Science 164:719, 1969; Cameron et al., Cli. Exp. Pharmacol.Physiol. 5:41, 1978), in which obesity is less severe than in thegenetic models and develops without hyperphagia, hyperinsulinemia andinsulin resistance, may also be examined. Finally, the streptozotocin(STZ) model for chemically-induced diabetes may be tested to examine theeffects of hyperglycemia in the absence of obesity. STZ-treated animalsare deficient in insulin and severely hyperglycemic (Coleman, Diabetes31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte,Jr. Eds. (Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990),pp. 299-340).

Modified FGF-21 polypeptides of the present disclosure can be evaluatedin an in vivo septic shock model in ob/ob mice. See U.S. PatentPublication No. 20050176631, which is incorporated by reference in itsentirety herein.

Methods for Analysis of ERK1/2 Phosphorylation Induced by FGF-21

ERK1/2 Phosphorylation Induced By FGF-21 (wild-type or modified FGF-21polypeptides including those linked to a PEG or other linker, polymer,or biologically active molecule) may be carried out by the followingexemplary methods:

Seed 293-stably transfected with human Klotho beta at 100,000 cells/well(DMEM+10% FBS) in a poly-Lys coated plate. The following day cells are100% confluent, media is aspirated off and replaced with fresh media andincubate overnight. After 24 hours cells are stimulated with theselected unmodified or modified FGF-21 or FGF-21 analog using asstandard FGF21WT. Each individual compound is prepared by diluting themin PBS/1% BSA. Cells are treated in triplicate for 10 min @ 37° C. inthe incubator. After 10 min incubation media is carefully aspirated offfrom each well and 40 ul of cold 1× Cell Signaling Lysis Buffercontaining protease/phosphatase inhibitors (PI cocktail, Na3VN4 andPMSF) are added to each well to produce cell lysates. 96 well/plate isplaced on ice for 20 minutes and then spun down at 4000 rpm for 10 min.Cell lysates are frozen down at −80° C. Later on each sample is thawedout and 10 ul of cell lysates is added to MSD treated plate coated withantibody capturing both the unphosphorylated and phosphorylated forms ofERK1/2. Incubation with primary antibody occurs for 2 hrs, then theplate is washed several times with specific buffer followed by additionof secondary antibody. After 1 hour the incubation plate is washed againseveral times. Buffer for reading is added to each well. The plate istransferred to MSD reading machine. The curve that is produced is basedon the anti-phosphorylated ERK1/2 reading units and EC50 is calculatedusing Sigma Plot. The fold loss of activity is calculated by dividingEC50 of the tested compound with the EC50 of the WT.

Determination of Pharmacokinetic Properties of Modified FGF-21Polypeptides in Rats

These exemplary methods may be used to measure the pharmacokineticproperties of native and modified FGF-21 compounds in catheterized rats.The pharmacokinetics of test articles are assayed by ELISA specific forhuman FGF-21 from serum samples obtained at specific time points afterdrug dosing.

Twelve (12) male Sprague-Dawley (SD) rats weighing approximately 250-275grams at study initiation may have had jugular vein catheters surgicallyplaced. Animals are in good condition and may have acclimated to thestudy location for at least 3 days prior to the start of the study. Ratsmay be weighed on the day of test article administration. Animals may behoused in standard, pathogen-free conditions with food and water adlibitum.

Compounds are administered subcutaneously, for example at 0.25 mg/kg.

Animals are weighed prior to administration of test article. Compoundsare formulated so as to be administered at 1×BW in μL. Subcutaneousadministration of test article is injected into the dorsal scapularregion. Animals may receive a single injection of test article (time=0).At selected time points, whole blood may be drawn from the animals,collected into SST microtainer collection tubes. Serum may be allowed toclot for 30 minutes prior to centrifugation. Serum may be transferred topolypropylene titer tubes, sealed with microstrips, and stored at −80degrees C. until analyzed by ELISA to determine unmodified or modifiedFGF-21 serum concentrations.

Each animal may be used for a complete PK time course. Approximately0.25 mL of whole blood may be drawn from the jugular vein cathetersImmediately after the blood collection, the catheters may be flushedwith 0.1 mL of saline. The following collection time points for animalsreceiving test article material are required selected but may bemodified based on the anticipated pharmacokinetic profile of the testarticles:

Pre-bleed, 1, 2, 4, 8, 24, 32 48, 56, 72, and 96 hours post-dose.Pharmacokinetic parameters are determined for each tested compound,which may include Lambda_z, Lambda_z_lower, Lambda_z_upper, HL_Lambda_z,Tmax, Cmax, C0, AUCINF_obs, Vz_obs, C1_obs, MRTINF_obs, and/or Vss_obs.

In Vivo Studies of Modified FGF-21 in ZDF Rats

Modified FGF-21, unmodified FGF-21, and buffer solution are administeredto mice or rats. The results show activity and half life of the modifiedFGF-21 polypeptides of the present disclosure compared to unmodifiedFGF-21.

WO 2005/091944 describes pharmacokinetic studies that can be performedwith FGF-21 compounds, such as the compounds of the present disclosure.A modified FGF-21 polypeptide of the present disclosure is administeredby intravenous or subcutaneous routes to mice. The animals are bledprior to and at time points after dosing. Plasma is collected from eachsample and analyzed by radioimmunoassay. Elimination half-life can becalculated and compared between modified FGF-21 polypeptides comprisingan internal deletion and/or a non-naturally encoded amino acid or fusionpartner, and wild-type FGF-21 or various forms of FGF-21 polypeptides ofthe present disclosure. Similarly, modified FGF-21 polypeptides of thepresent disclosure may be administered to cynomolgus monkeys. Theanimals are bled prior to and at time points after dosing. Plasma iscollected from each sample and analyzed by radioimmunoassay.

Polypeptides of the present disclosure may be administered to ZDF malerats (diabetic, fat rats; 8 weeks of age at beginning of study, CharlesRiver-GMI). Rats are fed Purina 5008 feed ad libitum. The following testgroups are set up: Saline; Insulin 4 U/day; unmodified or modifiedFGF-21, 500 ug/day Acute (Acute dosing group is dosed once and bled atT=0, 2, 4, 8, and 24 hours post dose); unmodified or modified FGF-21,100 ug/day; unmodified or modified FGF-21, 250 ug/day; unmodified ormodified FGF-21, 500 ug/day; unmodified or modified FGF-21(once/day) 500ug/ml; Lean Saline; Lean Insulin 4 U/day; Lean unmodified or modifiedFGF-21 500 ug/day. Lean groups represent non-diabetic, lean, ZDF rats.

Compounds are injected s.c. (b.i.d.), except for the second 500 ug/daygroup which receives one injection per day for the duration of the study(7 days). Control rats are injected with vehicle (PBS; 0.1 m1).Following 7 days of dosing, the animals are subjected to an oral glucosetolerance test. Blood for glucose and triglycerides are collected bytail clip bleeding without anesthetic. Modified FGF-21 polypeptides mayreduce plasma glucose levels in a dose-dependent manner. Also lean ZDFrats may not become hypoglycemic after exposure to modified FGF-21polypeptides of the present disclosure when compared to rats dosed withinsulin.

In Vivo Studies of Modified FGF-21 in Ob/Ob Obesity Model

The ob/ob mouse model is an animal model for hyperglycemia, insulinresistance, and obesity. Plasma glucose levels after treatment withunmodified or modified FGF-21 polypeptide compared to vehicle andinsulin control groups may be measured in ob/ob mice. In this obesitymodel, the test groups of male ob/ob mice (7 weeks old) are injectedwith vehicle alone (PBS), insulin (4 U/day), or unmodified or modifiedFGF-21 polypeptide (5 μg/day and 25 μg/day), subcutaneously (0.1 ml,b.i.d) for seven days. Blood is collected by tail clip bleeding on days1, 3, and 7, one hour after the first compound injection, and plasmaglucose levels are measured using a standard protocol. Modified FGF-21polypeptides of the present disclosure stimulate glucose uptake if theyreduce plasma glucose levels when compared to the vehicle control group.Triglyceride levels may be compared after treatment with modified FGF-21polypeptides of the present disclosure compared to other molecules. Thepolypeptide may be administered the mice via multiple doses, continuousinfusion, or a single dose, etc.

Pharmacokinetic Evaluation of Modified FGF21 Polypeptides:

The pharmacokinetic properties of modified FGF-21 with varying sites ofPEG conjugation are evaluated in rat. Other parameters studied are PEGMW, as well as dose of compound administered. The percentbioavailability is determined.

All animal experimentation is conducted under protocols approved by theInstitutional Animal Care and Use Committee. Male (175-300 g)Sprague-Dawley rats are obtained from Charles River Laboratories. Ratsare housed individually in cages in rooms with a 12-h light/dark cycleand acclimated to the vivarium for at least 3 days prior toexperimentation. Animals are provided access to certified Purina rodentchow 5001 and water ad libitum.

Catheters are surgically installed into the jugular vein for bloodcollection. Following successful catheter patency, animals are assignedto treatment groups prior to dosing. A single-dose of compound isadministered intravenously or subcutaneously in a dose volume of 1mL/kg. Compound dose concentrations are derived by dilution in PBS usingthe stock concentration as assigned in the Certificate of Release. Bloodsamples are collected at various time points via the indwelling catheterand placed into SST microfuge tubes. Serum is collected aftercentrifugation, and stored at −80° C. until analysis.

The assay for the quantification of modified FGF-21 in Sprague-Dawleyrat serum is performed as follows. Microplate wells are coated with goatanti-human FGF-21 IgG polyclonal antibody (PAb; RnD Systems, cloneAF2539) that is used as the capture reagent. Standard (STD) and qualitycontrol (QC) samples, both made by spiking unmodified or modified FGF-21into 100% Sprague Dawley rat serum, and study samples are loaded intothe wells after pre-treating 1:100 with I-Block buffer. The unmodifiedor modified FGF-21 in the STDs, QCs and study samples is captured by theimmobilized PAb. Unbound materials are removed by washing the wells.Biotin goat anti-human FGF-21 IgG PAb (RnD Systems, clone BAF2539) isadded to the wells followed by a wash step and the addition ofstreptavidin horseradish peroxidase (SA-HRP; RnD Systems, Catalog #DY998) for detection of the captured unmodified or modified FGF-21.After another washing step, tetramethylbenzidine (TMB, Kirkegaard PerryLaboratories) substrate solution is added to the wells. TMB reacts withthe peroxide in the presence of HRP and produces a colorimetric signalproportional to the amount of FGF-21 (wild-type or modified FGF-21)bound by the capture reagent in the initial step. The color developmentis stopped by the addition of 2N sulfuric acid and the intensity of thecolor (optical density, OD) is measured at 450 nm. The conversion of ODunits for the study samples and the QCs to concentration is achievedthrough a computer software mediated comparison to a standard curve onthe same plate, which is regressed according to a 5-parameter logisticregression model using SOFTmax Pro v5 data reduction package. Resultsare reported in ng/mL concentration units.

Concentrations may also be measured by a double antibody sandwich assayor other methods known to those skilled in the art. Concentrations arecalculated using a standard curve generated from the corresponding dosedcompound. Pharmacokinetic parameters are estimated using the modelingprogram WinNonlin (Pharsight, version 4.1). Noncompartmental analysisfor individual animal data with linear-up/log-down trapezoidalintegration is used, and concentration data is uniformly weighted.

XV. Administration and Pharmaceutical Compositions

Also provided herein are compositions comprising a therapeuticallyeffective amount of the modified FGF-21 polypeptide, described herein,and a pharmaceutically acceptable carrier or excipient. Such a carrieror excipient includes, but is not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and/or combinations thereof. Theformulation is made to suit the mode of administration. In general,methods of administering proteins are known to those of ordinary skillin the art and can be applied to administration of the polypeptides ofthe present disclosure.

For example, a modified FGF-21 polypeptide described herein may beadministered to a patient at a concentration of between about 0.1 and100 mg/kg of body weight of recipient patient. In an embodiment, amodified FGF-21 polypeptide described herein may be administered to apatient at a concentration of about 0.5-5 mg/kg of body weight ofrecipient patient. In another embodiment, a modified FGF-21 polypeptidedescribed herein may be administered to a recipient patient with afrequency of between once per day and once per two weeks, such as aboutonce or twice per week, once every two days, once every three days, onceevery four days, once every five days, or once every six days.

It is to be understood that the concentration of the modified FGF-21polypeptide administered to a given patient may be greater or lower thanthe exemplary administration concentrations set forth above.

Based upon the information provided in the present disclosure, a personof skill in the art would be able to determine an effective dosage andfrequency of administration through routine experimentation, for exampleguided by the disclosure herein and the teachings in Goodman, L. S.,Gilman, A., Brunton, L. L., Lazo, J. S., & Parker, K. L. (2006). Goodman& Gilman's the pharmacological basis of therapeutics. New York:McGraw-Hill; Howland, R. D., Mycek, M. J., Harvey, R. A., Champe, P. C.,& Mycek, M. J. (2006). Pharmacology. Lippincott's illustrated reviews.Philadelphia: Lippincott Williams & Wilkins; and Golan, D. E. (2008).Principles of pharmacology: the pathophysiologic basis of drug therapy.Philadelphia, Pa., (etc.): Lippincott Williams & Wilkins.

Average quantities of the modified FGF-21 may vary and in particularshould be based upon the recommendations and prescription of a qualifiedphysician. The exact amount of modified FGF-21 is a matter of preferencesubject to such factors as the exact type of condition being treated,the condition of the patient being treated, as well as the otheringredients in the composition. The present disclosure also provides foradministration of a therapeutically effective amount of another activeagent. The amount to be given may be readily determined by one ofordinary skill in the art.

A “pharmaceutical composition” refers to a chemical or biologicalcomposition suitable for administration to a mammal Such compositionsmay be specifically formulated for administration via one or more of anumber of routes, including but not limited to buccal, epicutaneous,epidural, inhalation, intraarterial, intracardial,intracerebroventricular, intradermal, intramuscular, intranasal,intraocular, intraperitoneal, intraspinal, intrathecal, intravenous,oral, parenteral, rectally via an enema or suppository, subcutaneous,subdermal, sublingual, transdermal, and transmucosal. In addition,administration can occur by means of injection, powder, liquid, gel,drops, or other means of administration.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents that arephysiologically compatible. In one embodiment, the carrier is suitablefor parenteral administration. Alternatively, the carrier can besuitable for intravenous, intraperitoneal, intramuscular, or sublingualadministration. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions.

In some embodiments, the pharmaceutical composition may be present inlyophilized form. The composition can be formulated as a solution,microemulsion, liposome, or other ordered structure suitable to highdrug concentration. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol), and suitable mixturesthereof. The invention further contemplates the inclusion of astabilizer in the pharmaceutical composition. The proper fluidity can bemaintained, for example, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants.

In many cases, it may be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition. By including an agent such as, monostearatesalts and gelatin, the absorption of the injectable compositions can beprolonged. Moreover, the polypeptide can be formulated in a time-releaseformulation, for example in a composition which includes a slow releasepolymer. The active compounds can be prepared with carriers that mayprotect the compound against rapid release, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations are knownto those skilled in the art.

Modified FGF-21 polypeptides and compositions of the present disclosuremay be administered by any conventional route suitable for proteins orpeptides, including, but not limited to parenterally, e.g. injectionsincluding, but not limited to, subcutaneously or intravenously or anyother form of injections or infusions. Polypeptide compositions can beadministered by a number of routes including, but not limited to oral,intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous,topical, sublingual, or rectal means. Compositions comprising modifiedFGF-21 may also be administered via liposomes. The modified FGF-21polypeptide, may be used alone or in combination with other suitablecomponents such as a pharmaceutical carrier. The modified FGF-21polypeptide may be used in combination with other agents, including butnot limited to, an oral anti-diabetic agent.

The term “anti-diabetic agent” shall mean any drug that is useful intreating, preventing, or otherwise reducing the severity of any glucosemetabolism disorder, or any complications thereof, including any of theconditions, disease, or complications described herein. Anti-diabeticagents include insulin, thiazolidinediones, sulfonylureas, benzoic acidderivatives, alpha-glucosidase inhibitors, or the like.

Current drugs or anti-diabetic agents used for managing diabetes and itsprecursor syndromes, such as insulin resistance, that are well-known inthe art include five classes of compounds: the biguanides, e.g.,metformin; thiazolidinediones, e.g., troglitazone; the sulfonylureas,e.g., tolbutamide and glyburide; benzoic acid derivatives, e.g.repaglinide; and glucosidase inhibitors. In addition to these agents, anumber of other therapies may be used in combination with the FGF-21polypeptides of the present disclosure to improve glucose control,including but not limited to DPP-4 inhibitors. The lead DPP-4 compoundstested in clinical trials include Vildagliptin (Galvus) (LAF237),Sitagliptin (Januvia), Saxagliptin and Alogliptin, Novartis compound1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidine (NVP DPP728)

Another category of anti-diabetic agents that is inhibitors of carnitinepalmitoyl-transferase I (CPT-I), such as etomoxir.

Other known anti-diabetic agents include insulin preparations (e.g.,animal insulin preparations extracted from pancreas of bovine and swine;human insulin preparations genetically synthesized using Escherichiacoli, yeast; zinc insulin; protamine zinc insulin; fragment orderivative of insulin (e.g., INS-1), oral insulin preparation), insulinsensitizers (e.g., pioglitazone or a salt thereof (preferablyhydrochloride), rosiglitazone or a salt thereof (preferably maleate),Netoglitazone, Rivoglitazone (CS-011), FK-614, the compound described inWO01/38325, Tesaglitazar (AZ-242), Ragaglitazar (N,N-622), Muraglitazar(BMS-298585), Edaglitazone (BM-13-1258), Metaglidasen (MBX-102),Naveglitazar (LY-519818), MX-6054, LY-510929, AMG-131(T-131), THR-0921),α-glucosidase inhibitors (e.g., voglibose, acarbose, miglitol,emiglitate etc.), biguanides (e.g., phenformin, metformin, buformin or asalt thereof (e.g., hydrochloride, fumarate, succinate)), insulinsecretagogues [sulfonylurea (e.g., tolbutamide, glibenclamide,gliclazide, chlorpropamide, tolazamide, acetohexamide, glyclopyramide,glimepiride, glipizide, glybuzole), repaglinide, nateglinide,mitiglinide or calcium salt hydrate thereof], dipeptidyl peptidase IVinhibitors (e.g., Vidagliptin (LAF237), P32/98, Sitagliptin (MK-431),P93/01, PT-100, Saxagliptin (BMS-477118), T-6666, TS-021), β3 agonists(e.g., AJ-9677), GPR40 agonists, glucagon-like polypeptides (I) (glp I),(glp2), or other diabetogenic peptide hormones, GLP-1 receptor agonists[e.g., GLP-1, GLP-1MR agent, N,N-2211, AC-2993 (exendin-4), BIM-51077,Aib(8,35)hGLP-1 (7,37)NH₂, CJC-[131], amylin agonists (e.g.,pramlintide), phosphotyrosine phosphatase inhibitors (e.g., sodiumvanadate), gluconeogenesis inhibitors (e.g., glycogen phosphorylaseinhibitors, glucose-6-phosphatase inhibitors, glucagon antagonists),SGLT2 (sodium-glucose cotransporter) inhibitors (e.g., T-1095),11β-hydroxysteroid dehydrogenase inhibitors (e.g., BVT-3498),adiponectin or agonists thereof, IKK inhibitors (e.g., AS-2868), leptinresistance improving drugs, somatostatin receptor agonists (compoundsdescribed in WO01/25228, WO03/42204, WO98/44921, WO98/45285, WO99/2273.5etc.), glucokinase activators (e.g., Ro-28-1675), GIP (Glucose-dependentinsulinotropic peptide) and the like can be mentioned.

The modified FGF-21 may also be made into aerosol formulations (i.e.,they can be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of modified FGF-21 can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials.

The dose administered to a patient, in the context of the presentdisclosure, is sufficient to have a beneficial therapeutic response inthe patient over time, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the formulation,and the activity, stability or serum half-life of the modified FGF-21polypeptide employed and the condition of the patient, as well as thebody weight or surface area of the patient to be treated.

The dose administered, for example, to a 70 kilogram patient, istypically in the range equivalent to dosages of currently-usedtherapeutic proteins, adjusted for the altered activity or serumhalf-life of the relevant composition.

For administration, formulations of the present disclosure areadministered at a rate determined by the LD-50 or ED-50 of the relevantformulation, and/or observation of any side-effects of the modifiedFGF-21 polypeptides at various concentrations, including but not limitedto, as applied to the mass and overall health of the patient.Administration can be accomplished via single or divided doses.

Modified FGF-21 polypeptides of the present disclosure can beadministered directly to a mammalian subject. Administration is by anyof the routes normally used for introducing FGF-21 polypeptide to asubject. Modified FGF-21 polypeptides of the present disclosure can beprepared in a mixture in a unit dosage injectable form (including butnot limited to, solution, suspension, or emulsion) with apharmaceutically acceptable carrier. Modified FGF-21 polypeptides of thepresent disclosure can also be administered by continuous infusion(using, including but not limited to, minipumps such as osmotic pumps),single bolus or slow-release depot formulations.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

The pharmaceutical compositions and formulations of the presentdisclosure may comprise a pharmaceutically acceptable carrier,excipient, or stabilizer.

Suitable carriers include but are not limited to, buffers containingsuccinate, phosphate, borate, HEPES, citrate, histidine, imidazole,acetate, bicarbonate, and other organic acids; antioxidants includingbut not limited to, ascorbic acid; low molecular weight polypeptidesincluding but not limited to those less than about 10 residues;proteins, including but not limited to, serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers including but not limited to,polyvinylpyrrolidone; amino acids including but not limited to, glycine,glutamine, asparagine, arginine, histidine or histidine derivatives,methionine, glutamate, or lysine; monosaccharides, disaccharides, andother carbohydrates, including but not limited to, trehalose, sucrose,glucose, mannose, or dextrins; chelating agents including but notlimited to, EDTA and edentate disodium; divalent metal ions includingbut not limited to, zinc, cobalt, or copper; sugar alcohols includingbut not limited to, mannitol or sorbitol; salt-forming counter ionsincluding but not limited to, sodium and sodium chloride; and/ornonionic surfactants including but not limited to Tween™ (including butnot limited to, Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20),Pluronics™ and other pluronic acids, including but not limited to, andother pluronic acids, including but not limited to, pluronic acid F68(poloxamer 188), or PEG. Suitable surfactants include for example butare not limited to polyethers based upon poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., (PEO-PPO-PEO),or poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide),i.e., (PPO-PEO-PPO), or a combination thereof. PEO-PPO-PEO andPPO-PEO-PPO are commercially available under the trade names Pluronics™,R-Pluronics™, Tetronics™ and R-Tetronics™ (BASF Wyandotte Corp.,Wyandotte, Mich.) and are further described in U.S. Pat. No. 4,820,352incorporated herein in its entirety by reference. Otherethylene/polypropylene block polymers may be suitable surfactants. Asurfactant or a combination of surfactants may be used to stabilizePEGylated modified FGF-21 against one or more stresses including but notlimited to stress that results from agitation. Some of the above may bereferred to as “bulking agents.” Some may also be referred to as“tonicity modifiers.” Antimicrobial preservatives may also be appliedfor product stability and antimicrobial effectiveness; suitablepreservatives include but are not limited to, benzyl alcohol,benzalkonium chloride, metacresol, methyl/propyl parabene, cresol, andphenol, or a combination thereof.

Modified FGF-21 polypeptides of the present disclosure, including thoselinked to water soluble polymers such as PEG can also be administered byor as part of sustained-release systems. Sustained-release compositionsinclude, including but not limited to, semi-permeable polymer matricesin the form of shaped articles, including but not limited to, films, ormicrocapsules. Sustained-release matrices include from biocompatiblematerials such as poly(2-hydroxyethyl methacrylate), ethylene vinylacetate (or poly-D-(−)-3-hydroxybutyric acid, polylactides (polylacticacid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (copolymers of lactic acid and glycolic acid)polyanhydrides, copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, poly(ortho)esters, polypeptides, hyaluronicacid, collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone.Sustained-release compositions also include a liposomally entrappedcompound. Liposomes containing the compound are prepared by methodsknown.

The dose administered to a patient in the context of the presentdisclosure should be sufficient to cause a beneficial response in thesubject over time. Generally, the total pharmaceutically effectiveamount of the modified FGF-21 polypeptide of the present disclosureadministered parenterally per dose is in the range of about 0.01μg/kg/day to about 100 μg/kg, or about 0.05 mg/kg to about 1 mg/kg, ofpatient body weight, although this is subject to therapeutic discretion.

In some embodiments, modified FGF-21 polypeptides of the presentdisclosure modulate the effect of an anti-diabetic agent. In anotherembodiment of the present disclosure, modified FGF-21 polypeptides maybe coadministered with an anti-diabetic agent. In another embodiment ofthe present disclosure, modified FGF-21 polypeptides may be administeredbefore treatment with an anti-diabetic agent. In another embodiment ofthe present disclosure, modified FGF-21 polypeptides may be administeredfollowing treatment with an anti-diabetic agent. In another embodimentof the present disclosure, modified FGF-21 polypeptides arecoadministered with metformin. In some embodiments, the modified FGF-21polypeptides of the present disclosure are coadministered with Klothobeta. In some embodiments, the modified FGF-21 polypeptides of thepresent disclosure are coadministered with Klotho beta that includes oneor more non-naturally encoded amino acids. In some embodiments, themodified FGF-21 polypeptides of the present disclosure arecoadministered with Klotho beta and an anti-diabetic agent. In someembodiments, the modified FGF-21 polypeptides of the present disclosureare coadministered with an anti-diabetic agent. In some embodiments,modified FGF-21 polypeptides of the present disclosure are used incombination with one or more of the following: Taurine, Alpha LipoicAcid, an extract of Mulberry, Chromium, Glutamine, Enicostemma littoraleBlume, Scoparia dulcis, an extract of Tarragon and Andrographispaniculata. In some embodiments, modified FGF-21 polypeptides of thepresent disclosure are used in combination with one or more of thefollowing: insulin preparations (e.g., animal insulin preparationsextracted from pancreas of bovine and swine; human insulin preparationsgenetically synthesized using Escherichia coli, yeast; zinc insulin;protamine zinc insulin; fragment or derivative of insulin (e.g., INS-1),oral insulin preparation), insulin sensitizers (e.g., pioglitazone or asalt thereof (preferably hydrochloride), rosiglitazone or a salt thereof(preferably maleate), Netoglitazone, Rivoglitazone (CS-011), FK-614, thecompound described in WO01/38325, Tesaglitazar (AZ-242), Ragaglitazar(N,N-622), Muraglitazar (BMS-298585), Edaglitazone (BM-13-1258),Metaglidasen (MBX-102), Naveglitazar (LY-519818), MX-6054, LY-510929,AMG-131(T-131), THR-0921), α-glucosidase inhibitors (e.g., voglibose,acarbose, miglitol, emiglitate etc.), biguanides (e.g., phenformin,metformin, buformin or a salt thereof (e.g., hydrochloride, fumarate,succinate)), insulin secretagogues [sulfonylurea (e.g., tolbutamide,glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide,glyclopyramide, glimepiride, glipizide, glybuzole), repaglinide,nateglinide, mitiglinide or calcium salt hydrate thereof], dipeptidylpeptidase IV inhibitors (e.g., Vidagliptin (LAF237), P32/98, Sitagliptin(MK-431), P93/01, PT-100, Saxagliptin (BMS-477118), T-6666, TS-021), β3agonists (e.g., AJ-9677), GPR40 agonists, glucagon-like polypeptides (I)(glp I), (glp2), or other diabetogenic peptide hormones, GLP-1 receptoragonists [e.g., GLP-1, GLP-1MR agent, N,N-2211, AC-2993 (exendin-4),BIM-51077, Aib(8,35)hGLP-1 (7,37)NH₂, CJC-[131], amylin agonists (e.g.,pramlintide), phosphotyrosine phosphatase inhibitors (e.g., sodiumvanadate), gluconeogenesis inhibitors (e.g., glycogen phosphorylaseinhibitors, glucose-6-phosphatase inhibitors, glucagon antagonists),SGLUT (sodium-glucose cotransporter) inhibitors (e.g., T-1095),11β-hydroxysteroid dehydrogenase inhibitors (e.g., BVT-3498),adiponectin or agonists thereof, IKK inhibitors (e.g., AS-2868), leptinresistance improving drugs, somatostatin receptor agonists (compoundsdescribed in WO01/25228, WO03/42204, WO98/44921, WO98/45285, WO99/22735etc.), glucokinase activators (e.g., Ro-28-1675), GIP (Glucose-dependentinsulinotropic peptide).

One way in which the therapeutic efficacy of the polypeptides andcombined therapies including the present disclosure's polypeptides maybe determined is through a reduction in patient HbA1c levels. In oneembodiment, polypeptides of the present disclosure lower HbA1c levels byat least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or at least50% change from HbA1c levels two months prior to beginning therapy withmodified FGF-21 polypeptides, from three months prior to beginningtherapy with modified FGF-21 polypeptides, or by percentage changes froma baseline. In another embodiment, polypeptides of the presentdisclosure administered to a patient also being treated with ananti-diabetic agent modulate the ability of the anti-diabetic agent tolower blood glucose.

In another embodiment, modified FGF-21 polypeptides of the presentdisclosure modulate the ability of Troglitazone to decrease insulinrequirements. Troglitazone used in combination with a polypeptide of thepresent disclosure may be used to delay or prevent Type 2 diabetes incertain embodiments of the present disclosure.

In one embodiment of the present disclosure, modified FGF-21polypeptides are coadministered with, or administered before or aftertreatment with, a sulfonylurea. In some embodiments of the presentdisclosure, treatment with a therapeutic dose of modified FGF-21polypeptides modulates serum glucose. In another embodiment, modifiedFGF-21 polypeptides of the present disclosure are administered withKlotho beta which modulates the effects of the polypeptides on bloodglucose. In another embodiment, modified FGF-21 polypeptides of thepresent disclosure are administered with Klotho beta which decreasesblood glucose more than use of modified FGF-21 polypeptides alone.

XVI. Therapeutic Uses of Modified FGF-21 Polypeptides of the PresentDisclosure

The modified FGF-21 polypeptides of the present disclosure may be usedto treat mammals suffering from non-insulin dependent Diabetes Mellitus(NIDDM: Type 2), insulin dependent diabetes (Type 1), as well asobesity, inadequate glucose clearance, hyperglycemia, hyperinsulinemia,and any other disease or condition that may be mediated by FGF-21.

Prader-Willi syndrome (P.W.S. or PWS) is a rare genetic disorder inwhich seven genes (or some subset thereof) on chromosome 15 (q 11-13)are deleted or unexpressed (chromosome 15q partial deletion) on thepaternal chromosome. Characteristic of PWS is low muscle tone, shortstature, incomplete sexual development, cognitive disabilities, problembehaviors, and a chronic feeling of hunger that can lead to excessiveeating and life-threatening obesity.

The present disclosure provides a method for treating a mammalexhibiting one or more of Type 1 diabetes, Type 2 diabetes, obesity,insulin resistance, hyperinsulinemia, glucose intolerance, orhyperglycemia, comprising administering to said mammal in need of suchtreatment a therapeutically effective amount of the modified FGF-21polypeptide of the present disclosure.

The method of treating may be sufficient to achieve in said mammal atleast one of the following modifications: reduction in body fat stores,decrease in insulin resistance, reduction of hyperinsulinemia, increasein glucose tolerance, and reduction of hyperglycemia.

The present disclosure also encompasses a method of reducing mortalityand morbidity in critically ill patients suffering from systemicinflammatory response syndrome (SIRS) associated with infectiousinsults, such a sepsis, pancreatitis, ischemia, multiple trauma andtissue injury, hemorrhagic shock, immune-mediated organ injury,respiratory distress, shock, renal failure, and multiple organdysfunction syndrome (MODS), as well as noninfectious pathologic causeswhich comprises administering to the critically ill patients atherapeutically effective amount of modified FGF-21.

In one embodiment, the disclosure provides compositions and methods withthe modified FGF-21 polypeptides disclosed herein in combination withanother agent for treating impaired glucose metabolism, such as insulinresistance, impaired insulin secretion or hyperglycemia. Such otheragents include, for example, one or more of sulfonylureas, PPAR-gammaagonists, GPL-1 receptor agonists, dipeptidyl peptidase IV inhibitor,amylin analogs, biguanides, dopamine D2 receptor agonists, meglitinides,alpha-glucosidase inhibitor, antidyslipidemic bile acid sequestrant,insulin, cytokine therapy, gene therapy, and antibody therapy. In someembodiments, the modified FGF-21 polypeptide disclosed herein may beadministered to a patient that does not achieve normoglycemia withadministration of another treatment, e.g., treatment with metformin,pioglitazone, a sulfonylurea, a glinide, an oral thiazolidinedione (TZD)such as pioglitazone, a glucagon-like peptide 1 (GLP-1) receptor agonistsuch as exenatide, a DPP4 inhibitor such as sitagliptin, vildagliptin,saxagliptin, alogliptin, linagliptin, or teneligliptin, or a combinationtherapy such as metformin and pioglitazone, metformin and asulfonylurea, metformin and a glinide, metformin and a TZD, metforminand pioglitazone, metformin and a GLP-1 receptor agonist, metformin andexenatide, sitagliptin and metformin, sitagliptin and simvastatin,vildagliptin and metformin, saxagliptin and metformin, alogliptin andpioglitazone, or linagliptin and metformin, or SGLT2 inhibitors such asdapagliflozin, canagliflozin, empagliflozin, ipragliflozin ortofogliflozin.

In some embodiment, the modified FGF-21 polypeptide disclosed herein maybe administered for prevention or treatment of obesity (e.g., a bodymass index of at least 25). Said the modified FGF-21 polypeptide may beadministered in combination with an anti-obesity agent such as orlistat,rimonabant, sibutramine, a peptide YY (PYY, a 36 amino acid peptide thatreduces appetite), a PYY analog, a CB-1 antagonist, rimonabant, aleptin, a leptin analog, or a phentermine.

In some embodiments, the modified FGF-21 polypeptide disclosed hereinmay be administered for prevention or treatment of Prader-Willisyndrome.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to those of ordinary skill in the artand are to be included within the spirit and purview of this applicationand scope of the appended claims. It is also to be understood that theterminology used herein is for the purpose of describing exemplaryembodiments only, and is not intended to limit the scope of the presentdisclosure, which are limited only by the appended claims. Allpublications, patents, patent applications, and/or other documents citedin this application are incorporated by reference in their entirety forall purposes to the same extent as if each individual publication,patent, patent application, and/or other document are individuallyindicated to be incorporated by reference for all purposes.

EXAMPLES

The following examples are offered to illustrate, but do not limit theclaimed invention.

Example 1 Expression and Purification of FGF-21 in E. Coli

Plasmids containing unmodified or modified FGF-21 expression constructsunder control of the T7 promoter are transformed into an E. coli strainsuch as the W3110 B2 strain of E. coli in which expression of the T7polymerase is under control of an arabinose-inducible promoter.Overnight bacterial cultures can be diluted 1:100 into shake flaskscontaining 2×YT culture media and grown at 37° C. to an OD₆₀₀ of ˜0.8.Protein expression can be induced by the addition of arabinose (0.2%final), and para-acetyl-phenylalanine (pAcF) to a final concentration of4 mM. Cultures may be incubated for a suitable duration and temperature,e.g., at 37 degrees C. for 4 hours. Cells can be pelleted andresuspended in B-PER lysis buffer (Pierce) 100 ul/OD/ml+10 ug/ml DNaseand incubated at 37° C. for 30 min. Cellular material can be removed bycentrifugation and the supernatant can be removed. The pellet can bere-suspended in an equal amount of SDS-PAGE protein loading buffer.Samples can be loaded on a 4-12% PAGE gel with MES and DTT. Methods forpurification of FGF-21 are known to those of ordinary skill in the artand purification can be confirmed by SDS-PAGE, Western Blot analyses,electrospray-ionization ion trap mass spectrometry and the like.

Cell paste is resuspended by mixing to a final 10% solid in 4° C.inclusion body (IB) Buffer I (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA;1% Triton X-100; 4° C.). Cells are lysed by passing resuspended materialthrough a micro fluidizer a total of two times, then it is centrifuged(10,000 g; 15 min; 4° C.) and the supernatant is decanted. The IB pelletis washed by resuspending in an additional volume of IB buffer I (50 mMTris pH 8.0; 100 mM NaCl; 1 mM EDTA; 1% Triton X-100; 4° C.) andresuspended material is passed through micro fluidizer a total of twotimes, then it is centrifuged (10,000 g; 15 min; 4° C.) and thesupernatant is decanted. The IB pellet is resuspended in one volume ofbuffer II (50 mM Tris pH 8.0; 100 mM NaCl; 1 mM EDTA; 4° C.), then it iscentrifuged (10,000 g; 15 min; 4° C.) and the supernatant is decanted.IB pellet is then resuspended in ½ volume of buffer II (50 mM Tris pH8.0; 100 mM NaCl; 1 mM EDTA; 4° C.). IB is aliquoted into appropriatecontainers, then it is centrifuged (10,000 g; 15 min; 4° C.) and thesupernatant is decanted. Inclusion bodies are solubilized (this is thepoint at which they could otherwise be stored at −80° C. until furtheruse.)

Inclusion bodies are solubilized to a final concentration between 10-15mg/mL in solubilization buffer (20 mM Tris, pH 8.0; 8M Urea; 10 mM β-ME)and incubated solubilized IB at room temperature under constant mixingfor 1 hour. Insoluble material is removed by filtration (0.45 μm PESfilter) and the protein concentration is adjusted (not always necessary)by dilution with additional solubilization buffer (when proteinconcentration is high).

Refolding can be effectuated by dilution to a final proteinconcentration of 0.5 mg/mL in 20 mM Tris, pH 8.0; 4° C. Allowed torefold for 18 to 24 hours at 4° C.

For purification, filtered refold reaction can be passed through a 0.45μM PES filter. Loaded material over a Q HP column (GE Healthcare)equilibrated in Buffer A (20 mM Tris, pH 7.5). Elution of unmodified ormodified FGF-21 with a linear gradient over 20CV to 100% Buffer B (20mMTris, pH 7.5; 250 mM NaCl). Monomeric FGF-21 can be thereby producedfrom pooled eluted fractions. Q HP pool is taken and buffer exchangedinto 20 mM Tris, pH 8.0; 2M urea; 1 mM EDTA. pH is dropped to 4.0 with50% glacial acetic acid. Sample are concentrated down to 4.0±1.0 mg/mL.To the sample is added a 12:1 molar excess PEG and a final concentrationof 1% Acetic Hydrazide, pH 4.0. The sample is incubate at 28° C. for48-72 hours. To the PEG reaction is then added a final of 50 mM Trisbase, and dilute 10 fold with RO water. Conductivity is verified to be<1 mS/cm and pH is between 8.0-9.0. Material is loaded over a Source 30Qcolumn (GE Healthcare) equilibrated in Buffer A (20 mM Tris, pH 8.0).PEG-FGF-21 is eluted with a linear gradient over 20CV to 100% B (20 mMTris, pH 8.0; 100 mM NaCl). PEG-FGF-21 is pooled and buffer exchangedinto 20 mM Tris, pH 7.4; 150 mM NaCl. PEG material is concentrated tobetween 1-2 mg/mL and filter sterilize using 0.22 μm PES filter.Material is stored at 4° C. For prolonged storage, flash freeze andstore at −80° C.

Example 2 Methods for Production of Modified FGF-21 PolypeptidesComprising a Non-Naturally Encoded Amino Acid

An introduced translation system that comprises an orthogonal tRNA(O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS) can be usedto express modified FGF-21 containing a non-naturally encoded aminoacid. The O-RS preferentially aminoacylates the O-tRNA with anon-naturally encoded amino acid. In turn the translation system insertsthe non-naturally encoded amino acid into FGF-21, in response to anencoded selector codon. Suitable O-RS and O-tRNA sequences are describedin WO 2006/068802 entitled “Compositions of Aminoacyl-tRNA Synthetaseand Uses Thereof” (E9; SEQ ID NO: 15) and WO 2007/021297 entitled“Compositions of tRNA and Uses Thereof” (F13; SEQ ID NO: 16), which areincorporated by reference in their entirety herein. Exemplary O-RS andO-tRNA sequences are included in Table 2 below.

TABLE 2 Sequence identifiers of exemplary orthogonal tRNA (O-tRNA) andorthogonal aminoacyl tRNA synthetase (O-RS) polypeptide andpolynucleotide sequences SEQ ID M. jannaschii mtRNA_(CUA) ^(Tyr) tRNANO: 17 SEQ ID HLAD03; an optimized amber suppressor tRNA tRNA NO: 18 SEQID HL325A; an optimized AGGA frameshift suppressor tRNA NO: 19 tRNA SEQID Aminoacyl tRNA synthetase for the incorporation of RS NO: 20p-azido-L-phenylalanine p-Az-PheRS(6) SEQ ID Aminoacyl tRNA synthetasefor the incorporation RS NO: 21 of p-benzoyl-L-phenylalanine p-BpaRS(1)SEQ ID Aminoacyl tRNA synthetase for the incorporation RS NO: 22 ofpropargyl-phenylalanine Propargyl-PheRS SEQ ID Aminoacyl tRNA synthetasefor the incorporation RS NO: 23 of propargyl-phenylalaninePropargyl-PheRS SEQ ID Aminoacyl tRNA synthetase for the incorporationRS NO: 24 of propargyl-phenylalanine Propargyl-PheRS SEQ ID AminoacyltRNA synthetase for the incorporation RS NO: 25 of p-azido-phenylalaninep-Az-PheRS(1) SEQ ID Aminoacyl tRNA synthetase for the incorporation RSNO: 26 of p-azido-phenylalanine p-Az-PheRS(3) SEQ ID Aminoacyl tRNAsynthetase for the incorporation RS NO: 27 of p-azido-phenylalaninep-Az-PheRS(4) SEQ ID Aminoacyl tRNA synthetase for the incorporation RSNO: 28 of p-azido-phenylalanine p-Az-PheRS(2) SEQ ID Aminoacyl tRNAsynthetase for the incorporation RS NO: 29 of p-acetyl-phenylalanine(LW1) SEQ ID Aminoacyl tRNA synthetase for the incorporation RS NO: 30of p-acetyl-phenylalanine (LW5) SEQ ID Aminoacyl tRNA synthetase for theincorporation RS NO: 31 of p-acetyl-phenylalanine (LW6) SEQ ID AminoacyltRNA synthetase for the incorporation RS NO: 32 of p-azido-phenylalanine(AzPheRS-5) SEQ ID Aminoacyl tRNA synthetase for the incorporation RSNO: 33 of p-azido-phenylalanine (AzPheRS-6)

TABLE 3 Exemplary FGF-21, orthogonal tRNA (O-tRNA)and an orthogonal aminoacyl tRNA synthetase(O-RS) polypeptide and polynucleotide sequences SEQ ID # Sequence Name 1 Amino acid sequence of FGF-21 without leader (P-form)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS  2 Amino acid sequence of FGF-21 without leader(P-form)-His tagged MHHHHHHSGGHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS  3Amino acid sequence of FGF-21 with leader (P-form)-leader with 3 leucines (“209 amino acid P-form” or  “P-form”)MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS  4Amino acid sequence of FGF-21 with leader (P-form)-leader with two leucines MDSDETGFEHSGLWVSVLAGLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS  5Amino acid sequence of FGF-21 without leader (L-form)His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe GlyGly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp AlaGln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp GlyThr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser LeuLeu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln IleLeu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg ProAsp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro GluAla Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly TyrAsn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu HisLeu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala ProArg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu ProPro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro GlnPro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met ValGly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser  6Amino acid sequence of FGF-21 with leader (L-form)-leader with 3 leucines (209 amino acid L-form)Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly LeuTrp Val Ser Val Leu Ala Gly Leu Leu Leu Gly Ala CysGln Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu GlnPhe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr AspAsp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg GluAsp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro GluSer Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val IleGln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys GlnArg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe AspPro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu AspGly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu ProLeu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp ProAla Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro GlyLeu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu AlaPro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu SerMet Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser  7Amino acid sequence of FGF-21 with leader (L-form)-leader with 2 leucines (208 amino acid L-form)Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly LeuTrp Val Ser Val Leu Ala Gly Leu Leu Gly Ala Cys GlnAla His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln PheGly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp AspAla Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu AspGly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu SerLeu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile GlnIle Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln ArgPro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp ProGlu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp GlyTyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro LeuHis Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro AlaPro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly LeuPro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala ProGln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser MetVal Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser  8Nucleotide Sequence for FGF-21 without leader (P-form)CACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCC CAGCTACGCTTCCTGA 9 Nucleotide Sequence for FGF-21 without leader (P-form)-His taggedATGCATCATCATCATCATCATAGCGGCGGCCACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCTTCCTGA 10Nucleotide Sequence for FGF-21 with leader (P-form)-leader with 3 leucinesATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCTTCCTGA 11Nucleotide Sequence for FGF-21 with leader (P-form)-leader with 2 leucinesATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTGCTGGCTGGTCTTCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCTTCCTGA 12Nucleotide Sequence for FGF-21 without leader (L-form)CACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCA GCTACGCTTCCTGA 13Nucleotide Sequence for FGF-21 with leader (L-form)-leader with 3 leucinesATG GAC TCG GAC GAG ACC GGG TTC GAG CAC TCA GGA CTG TGG GTTTCT GTG CTG GCT GGT CTT CTG CTG GGA GCC TGC CAG GCA CAC CCCATC CCT GAC TCC AGT CCT CTC CTG CAA TTC GGG GGC CAA GTC CGGCAG CGG TAC CTC TAC ACA GAT GAT GCC CAG CAG ACA GAA GCC CACCTG GAG ATC AGG GAG GAT GGG ACG GTG GGG GGC GCT GCT GAC CAGAGC CCC GAA AGT CTC CTG CAG CTG AAA GCC TTG AAG CCG GGA GTTATT CAA ATC TTG GGA GTC AAG ACA TCC AGG TTC CTG TGC CAG CGGCCA GAT GGG GCC CTG TAT GGA TCG CTC CAC TTT GAC CCT GAG GCCTGC AGC TTC CGG GAG CTG CTT CTT GAG GAC GGA TAC AAT GTT TACCAG TCC GAA GCC CAC GGC CTC CCG CTG CAC CTG CCA GGG AAC AAGTCC CCA CAC CGG GAC CCT GCA CCC CGA GGA CCA GCT CGC TTC CTGCCA CTA CCA GGC CTG CCC CCC GCA CTC CCG GAG CCA CCC GGA ATCCTG GCC CCC CAG CCC CCC GAT GTG GGC TCC TCG GAC CCT CTG AGCATG GTG GGA CCT TCC CAG GGC CGA AGC CCC AGC TAC GCT TCC TGA 14Nucleotide Sequence for FGF-21 with leader (L-form)-leader with 2 leucinesATG GAC TCG GAC GAG ACC GGG TTC GAG CAC TCA GGA CTG TGG GTTTCT GTG CTG GCT GGT CTT CTG GGA GCC TGC CAG GCA CAC CCC ATCCCT GAC TCC AGT CCT CTC CTG CAA TTC GGG GGC CAA GTC CGG CAGCGG TAC CTC TAC ACA GAT GAT GCC CAG CAG ACA GAA GCC CAC CTGGAG ATC AGG GAG GAT GGG ACG GTG GGG GGC GCT GCT GAC CAG AGCCCC GAA AGT CTC CTG CAG CTG AAA GCC TTG AAG CCG GGA GTT ATTCAA ATC TTG GGA GTC AAG ACA TCC AGG TTC CTG TGC CAG CGG CCAGAT GGG GCC CTG TAT GGA TCG CTC CAC TTT GAC CCT GAG GCC TGCAGC TTC CGG GAG CTG CTT CTT GAG GAC GGA TAC AAT GTT TAC CAGTCC GAA GCC CAC GGC CTC CCG CTG CAC CTG CCA GGG AAC AAG TCCCCA CAC CGG GAC CCT GCA CCC CGA GGA CCA GCT CGC TTC CTG CCACTA CCA GGC CTG CCC CCC GCA CTC CCG GAG CCA CCC GGA ATC CTGGCC CCC CAG CCC CCC GAT GTG GGC TCC TCG GAC CCT CTG AGC ATGGTG GGA CCT TCC CAG GGC CGA AGC CCC AGC TAC GCT TCC TGA 34Amino acid sequence of FGF-21 (Rattus norvegicus- ref|NP_570108.1|[18543365])Met Asp Trp Met Lys Ser Arg Val Gly Ala Pro Gly LeuTrp Val Cys Leu Leu Leu Pro Val Phe Leu Leu Gly ValCys Glu Ala Tyr Pro Ile Ser Asp Ser Ser Pro Leu LeuGln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr ThrAsp Asp Asp Gln Asp Thr Glu Ala His Leu Glu Ile ArgGlu Asp Gly Thr Val Val Gly Thr Ala His Arg Ser ProGlu Ser Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly ValIle Gln Ile Leu Gly Val Lys Ala Ser Arg Phe Leu CysGln Gln Pro Asp Gly Thr Leu Tyr Gly Ser Pro His PheAsp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu LysAsp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly LeuPro Leu Arg Leu Pro Gln Lys Asp Ser Gln Asp Pro AlaThr Arg Gly Pro Val Arg Phe Leu Pro Met Pro Gly LeuPro His Glu Pro Gln Glu Gln Pro Gly Val Leu Pro ProGlu Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser MetVal Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr Ala Ser 35Amino acid sequence of FGF-21 (Mus musculus- ref|NP_064397.1|[9910218])Met Glu Trp Met Arg Ser Arg Val Gly Thr Leu Gly LeuTrp Val Arg Leu Leu Leu Ala Val Phe Leu Leu Gly ValTyr Gln Ala Tyr Pro Ile Pro Asp Ser Ser Pro Leu LeuGln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr ThrAsp Asp Asp Gln Asp Thr Glu Ala His Leu Glu Ile ArgGlu Asp Gly Thr Val Val Gly Ala Ala His Arg Ser ProGlu Ser Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly ValIle Gln Ile Leu Gly Val Lys Ala Ser Arg Phe Leu CysGln Gln Pro Asp Gly Ala Leu Tyr Gly Ser Pro His PheAsp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu GluAsp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly LeuPro Leu Arg Leu Pro Gln Lys Asp Ser Pro Asn Gln AspAla Thr Ser Trp Gly Pro Val Arg Phe Leu Pro Met ProGly Leu Leu His Glu Pro Gln Asp Gln Ala Gly Phe LeuPro Pro Glu Pro Pro Asp Val Gly Ser Ser Asp Pro LeuSer Met Val Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr Ala Ser 36Amino acid sequence of FGF-21 (Danio rerio-ref|NP_001038789.1|[113671792])Met Leu Phe Ala Cys Phe Phe Ile Phe Phe Ala Leu PhePro His Leu Arg Trp Cys Met Tyr Val Pro Ala Gln AsnVal Leu Leu Gln Phe Gly Thr Gln Val Arg Glu Arg LeuLeu Tyr Thr Asp Gly Leu Phe Leu Glu Met Asn Pro AspGly Ser Val Lys Gly Ser Pro Glu Lys Asn Leu Asn CysVal Leu Glu Leu Arg Ser Val Lys Ala Gly Glu Thr ValIle Gln Ser Ala Ala Thr Ser Leu Tyr Leu Cys Val AspAsp Gln Asp Lys Leu Lys Gly Gln His His Tyr Ser AlaLeu Asp Cys Thr Phe Gln Glu Leu Leu Leu Asp Gly TyrSer Phe Phe Leu Ser Pro His Thr Asn Leu Pro Val SerLeu Leu Ser Lys Arg Gln Lys His Gly Asn Pro Leu SerArg Phe Leu Pro Val Ser Arg Ala Glu Asp Ser Arg ThrGln Glu Val Lys Gln Tyr Ile Gln Asp Ile Asn Leu AspSer Asp Asp Pro Leu Gly Met Gly His Arg Ser His LeuGln Thr Val Phe Ser Pro Ser Leu His Thr Lys Lys 37Amino acid sequence of Klotho beta (Homo sapiens- ref|NP_783864.1|[28376633])MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVTGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS 38Amino acid sequence of Klotho beta (Mus musculus- refNP_112457.1 GI:13626032)MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAVTGFSGDGKAIWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDCTICSFLVEKKPLIFFGCCFISTLAVLLSITVFHHQKRRKFQKARNLQNIPLKKGHSRVFS 39 OmpA nucleotide leader sequenceatgaaaaaaactgctatcgcgatcgctgtagctctggctggtttcgcgaccgt agctaacgct 40OmpA amino acid leader sequenceM K K T A I A I A V A L A G F A T V A N A 41MalE nucleotide leader sequenceatgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgcc 42 MalE amino acid leader sequenceM K I K T G A R I L A L S A L T T M M F S A S A L A 43StII nucleotide leader sequenceatgaaaaagaatatcgcatttcttcttgcatctatgttcgttttttctattgc tacaaatgcctatgca44 StII amino acid leader sequenceM K K N I A F L L A S M F V F S I A T N A Y A

The transformation of E. coli with plasmids containing the modifiedFGF-21 gene and the orthogonal aminoacyl tRNA synthetase/tRNA pair(specific for the desired non-naturally encoded amino acid) allows thesite-specific incorporation of non-naturally encoded amino acid into themodified FGF-21 polypeptide.

Wild type mature FGF-21 can be amplified by PCR from a cDNA synthesisreaction, e.g., derived from healthy human liver polyA+mRNA (Biochain)using standard protocols and cloned into a vector such as pET30 (e.g.,utilizing the NcoI-BamHI cleavage sites). Following optionally sequenceconfirmation, FGF-21 (which may include a purification tag such as anN-terminal HHHHHHSGG sequence) can be subcloned. For example, FGF-21 canbe subcloned into a suppression vector containing an amber suppressortyrosyl tRNA^(Tyr/CUA) from Methanococcus jannaschii (Mj tRNA^(Tyr/CUA))and linked to a suitable promoter, e.g., under constitutive control of asynthetic promoter derived from the E. coli lipoprotein promotersequence (Miller, J. H., Gene, 1986), as well as well as the orthogonaltyrosyl-tRNA-synthetase (MjTyrRS) under control of the E. coli GlnRSpromoter. Expression of unmodified or modified FGF-21 can be undercontrol of the T7 promoter. Amber mutations can be introduced usingstandard quick change mutation protocols (Stratagene; La Jolla, Calif.).Constructs may be sequence verified.

Two distinct non-naturally encoded amino acids may be introduced intomodified FGF-21 polypeptides, by introducing a selector codon at twodistinct sites within the nucleic acid

Suppression with Para-Acetyl-Phenylalanine (pAcF)

Plasmids containing modified FGF-21 expression constructs and O-tRNA andO-RS expression constructs (which may be in the same or differentplasmids or stably transfected into the strain) can be transformed intothe W3110 B2 strain of E. coli in which expression of the T7 polymeraseis under control of an arabinose-inducible promoter. Overnight bacterialcultures can be diluted 1:100 into shake flasks containing 2×YT culturemedia and grown at 37° C. to an OD₆₀₀ of ˜0.8. Protein expression can beinduced by the addition of arabinose (0.2% final), andpara-acetyl-phenylalanine (pAcF) to a final concentration of 4 mM.Cultures may be incubated for a suitable duration and temperature, e.g.,at 37 degrees C. for 4 hours. Cells can be pelleted and resuspended inB-PER lysis buffer (Pierce) 100 ul/OD/ml+10 ug/ml DNase and incubated at37° C. for 30 min. Cellular material can be removed by centrifugationand the supernatant can be removed. The pellet can be re-suspended in anequal amount of SDS-PAGE protein loading buffer. Samples can be loadedon a 4-12% PAGE gel with MES and DTT. Methods for purification of FGF-21are known to those of ordinary skill in the art and purification can beconfirmed by SDS-PAGE, Western Blot analyses, electrospray-ionizationion trap mass spectrometry and the like.

His-tagged or non-His-tagged mutant FGF-21 proteins can be purifiedusing methods known to those of ordinary skill in the art. For example,the ProBond Nickel-Chelating Resin (Invitrogen, Carlsbad, Calif.) may beused via the standard His-tagged protein purification proceduresprovided by the manufacturer.

Example 3 Introduction of a Carbonyl-Containing Amino Acid into aModified FGF-21 Polypeptide and Subsequent Reaction with anAminooxy-Containing PEG

The following exemplary method may be used for the generation of amodified FGF-21 polypeptide linked to a half-life extending moiety. Themethod incorporates a ketone-containing non-naturally encoded amino acidthat is subsequently reacted with a half-life extending moiety such asan aminooxy-containing PEG having a molecular weight of approximately30,000 Da. A selected residue of FGF-21 is substituted with anon-naturally encoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into FGF-21 may be SEQ ID NO: 1 (FGF-21), SEQ IDNO: 16 or 17 (muttRNA, M. jannaschii mtRNA_(CUA) ^(Tyr)), 15, 29, 30 or31 (TyrRS LW1, 5, or 6), or any modified FGF-21 polypeptide describedherein.

Once modified, the FGF-21 polypeptide comprising the carbonyl-containingamino acid is reacted with an aminooxy-containing PEG derivative of theform:

R-PEG(N)—O—(CH₂)_(n)—O—NH₂

where R is methyl, n is 3 and N is a selected molecular weight, e.g.,approximately 30,000 Da.

Alternatively, the ketone-containing non-naturally encoded amino acidcan be liked to a PEG reagent having the following structure:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—O—NH₂

where R=methyl, n=4 and N is a selected molecular weight, e.g.,approximately 30,000 Da.

The purified modified FGF-21 containing p-acetylphenylalanine dissolvedat 10 mg/mL in 25 mM MES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mMHepes (Sigma Chemical, St. Louis, Mo.) pH 7.0, or in 10 mM SodiumAcetate (Sigma Chemical, St. Louis, Mo.) pH 4.5, is reacted with a 10 to100-fold excess of aminooxy-containing PEG, and then stirred for 10-16hours at room temperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp475). The PEG-FGF-21 is then diluted into appropriate buffer forimmediate purification and analysis.

Example 4 Production of Modified FGF-21 Polypeptides

Polynucleotides encoding each of the modified FGF-21 polypeptides shownin FIG. 1A-B were produced. DNA codon usage in the polynucleotidesequences was optimized for E. coli expression using standard methodsknown in the art (SEQ ID NOs: 317 and 318 are exemplary polynucleotidesequences that encode Compound 2 and Pegylated Compound 2,respectively). If a connector peptide was present, its coding sequencewas selected based upon the standard genetic code, e.g., GGU, GGC, GGA,or GGG for glycine, UCU, UCC, UCA, UCG, AGU, or AGC for serine, CAU orCAC for histidine, etc. The modified FGF-21 polypeptides were expressedin E. coli and purified (exemplary methods are described herein).Modified FGF-21 polypeptides containing the non-naturally encoded aminoacid para-acetyl-phenylalanine (abbreviated pACF or pAF) were produced(exemplary methods are provided in Example 2). In brief, the encodingpolynucleotide was further modified to incorporate a selector codon atthe corresponding position in the polynucleotide sequence in order toencode the pACF, and were expressed in a cell engineered to express anorthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase(O-RS), such that the selector codon directed inclusion of the pACF atthe selected position. The pACF was then linked to a poly(ethyleneglycol) (PEG) having an average molecular weight of about 30 kDa(exemplary methods are provided in Example 3).

Compound 2 Coding Sequence (SEQ ID NO: 317) M   H   P   I   P   D   S   S   P   L   L   Q   F   G   G   Q   V   R   Q   R  1ATG CAT CCT ATT CCT GAT TCT TCT CCT CTG CTG CAA TTT GGG GGT CAG GTG CGC CAA CGT Y   L   Y   T   D   D   A   Q   Q   T   E   A   H   L   E   I   R   E   D   G 61TAC CTG TAC ACC GAC GAT GCG CAA CAG ACT GAG GCT CAC CTG GAG ATC CGT GAG GAC GGG T   V   G   G   A   A   D   Q   S   P   E   S   L   L   Q   L   K   A   L   K121ACT GTC GGA GGG GCT GCC GAT CAA TCC CCA GAG TCA CTG CTG CAA CTG AAA GCC CTG AAG P   G   V   I   Q   I   L   G   V   K   T   S   R   F   L   C   Q   R   P   D181CCT GGG GTC ATT CAG ATC CTG GGC GTA AAG ACG AGT CGT TTC CTG TGC CAA CGT CCT GAC G   A   L   Y   G   S   L   H   F   D   P   E   A   C   S   F   R   E   L   L241GGG GCA CTG TAT GGC TCG CTG CAT TTT GAT CCT GAG GCT TGT AGT TTT CGC GAA CTG CTG L   E   D   G   Y   N   V   Y   Q   S   E   A   H   G   L   P   L   H   L   G301CTG GAA GAT GGT TAC AAT GTG TAT CAG AGT GAA GCA CAC GGT CTG CCT CTG CAC CTG GGT S   G   R   G   P   A   R   F   L   P   L   P   G   L   P   P   A   P   P   E361TCT GGT CGT GGT CCG GCG CGT TTT CTG CCA CTG CCT GGC CTG CCT CCA GCA CCA CCT GAA P   P   G   I   L   A   P   Q   P   P   D   V   G   S   S   D   P   L   S   M421CCA CCG GGT ATT CTG GCT CCG CAA CCT CCA GAC GTC GGG AGT TCA GAT CCT CTG TCG ATG V   E   P   S   Q   G   R   S   P   S   Y   A   S 481GTA GAA CCG TCA CAA GGT CGC TCT CCT AGT TAC GCG TCAPEGylated-Compound 2 Coding Sequence (SEQ ID NO: 318) M   H   P   I   P   D   S   S   P   L   L   Q   F   G   G   Q   V   R   Q   R  1ATG CAT CCT ATT CCT GAT TCT TCT CCT CTG CTG CAA TTT GGG GGT CAG GTG CGC CAA CGT Y   L   Y   T   D   D   A   Q   Q   T   E   A   H   L   E   I   R   E   D   G 61TAC CTG TAC ACC GAC GAT GCG CAA CAG ACT GAG GCT CAC CTG GAG ATC CGT GAG GAC GGG T   V   G   G   A   A   D   Q   S   P   E   S   L   L   Q   L   K   A   L   K121ACT GTC GGA GGG GCT GCC GAT CAA TCC CCA GAG TCA CTG CTG CAA CTG AAA GCC CTG AAG P   G   V   I   Q   I   L   G   V   K   T   S   R   F   L   C   Q   R   P   D181CCT GGG GTC ATT CAG ATC CTG GGC GTA AAG ACG AGT CGT TTC CTG TGC CAA CGT CCT GAC G   A   L   Y   G   S   L   H   F   D   P   E   A   C   S   F   R   E   L   L241GGG GCA CTG TAT GGC TCG CTG CAT TTT GAT CCT GAG GCT TGT AGT TTT CGC GAA CTG CTG L   E   D   G   Y   N   V   Y  pAF  S   E   A   H   G   L   P   L   H   L   G301CTG GAA GAT GGT TAC AAT GTG TAT TAG AGT GAA GCA CAC GGT CTG CCT CTG CAT CTG GGC S   G   R   G   P   A   R   F   L   P   L   P   G   L   P   P   A   P   P   E361TCC GGC CGC GGT CCG GCC CGT TTT CTG CCA CTG CCT GGC CTG CCT CCA GCA CCA CCT GAA P   P   G   I   L   A   P   Q   P   P   D   V   G   S   S   D   P   L   S   M421CCA CCG GGT ATT CTG GCT CCG CAA CCT CCA GAC GTC GGG AGT TCA GAT CCT CTG TCG ATG V   E   P   S   Q   G   R   S   P   S   Y   A   S 481GTA GAA CCG TCA CAA GGT CGC TCT CCT AGT TAC GCG TCA

As detailed in the examples that follow, the modified FGF-21polypeptides were further characterized, including testing for in vitrobiological activity, stability, propensity for deamidation and aggregateformation, resistance to proteolysis in vivo, immunogenicity,pharmacokinetics, and biological activity in vivo in a diabetes model.

The full polypeptide sequence of the modified FGF-21 polypeptidesproduced (whose partial sequences are shown in FIG. 1A-B) are asfollows:

Compound 1 (SEQ ID NO: 101) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSY AS Compound 2 (SEQ ID NO: 102)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGI LAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 3 (SEQ ID NO: 103) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSY AS Compound 4 (SEQ ID NO: 104)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGKKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSY AS Compound 5 (SEQ ID NO: 105)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGDKSRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 6 (SEQ ID NO: 106)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGHKSRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 7 (SEQ ID NO: 107)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGDKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSY AS Compound 8 (SEQ ID NO: 108)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGARFLPLPGLPPAPPEPPGILAP QPPDVGSSDPLSMVEPSQGRSPSYASCompound 9 (SEQ ID NO: 109) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGQKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSY AS Compound 10 (SEQ ID NO: 110)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGGPARFLPLPGLPPAPPEPPGIL APQPPDVGSSDPLSMVEPSQGRSPSYASCompound 11 (SEQ ID NO: 111) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGHRDPAPRGPARFLPLPGLPPAP PEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 12 (SEQ ID NO: 112) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPHHSGRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 13 (SEQ ID NO: 113)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGKDSQDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 14 (SEQ ID NO: 114)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGHKSRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS Compound 15 (SEQ ID NO: 115)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGHKSRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGREPSYAS Compound 16 (SEQ ID NO: 116)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGHKSRDPAPRGPARFLPLPGLPPA PPEPPGILAPQPPDVGSSDPLSMVPSQGRSPSYASCompound 17 (SEQ ID NO: 117) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGHKSRDPAPRGPARFLPLPGLPPA PPEPPGILAPQPPDVGSSDPLSMVGSQGRSPSYASCompound 18 (SEQ ID NO: 118) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLP PAPPEPPGILAPQPPDVGSSDPLSMVEPCompound 19 (SEQ ID NO: 119) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 20 (SEQ ID NO: 120)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGGHRDPAPRGPARFLPLPGLPPAPP EPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 21 (SEQ ID NO: 121) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRDPAPRGPARFLPLPGLPPAPP EPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 22 (SEQ ID NO: 122) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLSGGPAPRGPARFLPLPGLPPAPPEP PGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 23 (SEQ ID NO: 123) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGGGPARFLPLPGLPPAPPEPPGILA PQPPDVGSSDPLSMVEPSQGRSPSYASCompound 24 (SEQ ID NO: 124) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGGRFLPLPGLPPAPPEPPGILAPQ PPDVGSSDPLSMVEPSQGRSPSYASCompound 25 (SEQ ID NO: 125) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPSGGRFLPLPGLPPAPPEPPGILAP QPPDVGSSDPLSMVEPSQGRSPSYASCompound 26 (SEQ ID NO: 126) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHSGGPAPRGPARFLPLPGLPPAPPEPP GILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 27 (SEQ ID NO: 127) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHGSGGPARFLPLPGLPPAPPEPPGILA PQPPDVGSSDPLSMVEPSQGRSPSYASCompound 28 (SEQ ID NO: 128) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPHGGRFLPLPGLPPAPPEPPGILAPQPPD VGSSDPLSMVEPSQGRSPSYAS Compound 29(SEQ ID NO: 129) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPHSGGRFLPLPGLPPAPPEPPGILAPQPP DVGSSDPLSMVEPSQGRSPSYAS Compound 30(SEQ ID NO: 130) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPHGSGRFLPLPGLPPAPPEPPGILAPQPP DVGSSDPLSMVEPSQGRSPSYAS Compound 31(SEQ ID NO: 131) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKISRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGGPARFLPLPGLPPAPPEPPGIL APQPPDVGSSDPLSMVTPSQGRSPSYASCompound 32 (SEQ ID NO: 132) MHHHHHHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQG RSPSYAS Pegylated Compound 1(SEQ ID NO: 201) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGR SPSYAS Pegylated Compound 2(SEQ ID NO: 202) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLGSGRGPARFLPLPGLPPAPPE PPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASPegylated Compound 5 (SEQ ID NO: 205)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLPGDKSRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSP SYAS Pegylated Compound 6(SEQ ID NO: 206) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLPGHKSRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSP SYAS Pegylated Compound 10(SEQ ID NO: 210) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLGSGGPARFLPLPGLPPAPPEP PGILAPQPPDVGSSDPLSMVEPSQGRSPSYASPegylated Compound 11 (SEQ ID NO: 211)MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLGSGHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPS YAS Pegylated Compound 12(SEQ ID NO: 212) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLPHHSGRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSP SYAS Pegylated Compound 19(SEQ ID NO: 219) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLGSGPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSP SYAS Pegylated Compound 20(SEQ ID NO: 220) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLGGHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSY AS Pegylated Compound 21(SEQ ID NO: 221) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLGSGRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSY AS Pegylated Compound 22(SEQ ID NO: 222) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLSGGPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Pegylated Compound 23(SEQ ID NO: 223) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLGGGPARFLPLPGLPPAPPEPP GILAPQPPDVGSSDPLSMVEPSQGRSPSYAS

Additional modified FGF-21 polypeptides were produced as fusionproteins. Some of the modified FGF-21 polypeptides contained thesequence of Compound 2 (SEQ ID NO:102), with or without the N-terminalmethionine, fused to one or more fusion partners, such as an Fc domainor fragment thereof, a PKE Adnectin (“PKE”), or modified or unmodifiedhuman serum albumin (“HSA”) sequence and optionally a connectingpeptide. Other modified FGF-21 polypeptides were based upon a wild-typesequence of Compound 1 (with or without the N-terminal methionine).Different PKE Adnectin sequence forms were included. These are referredto as “PKE(1)” or “PKEI” and “PKE(2)” or PKEII. The amino acid sequenceof PKE(1) wasGVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQ (SEQ ID NO:319). The amino acid sequenceof PKE(2) wasGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP (SEQ ID NO:320). When PKE(1) orPKE(2) was included at the N-terminus of the fusion protein, thesequence expressed in E. coli included an N-terminal methionine, whichwas expected to be cleaved by a met-exopeptidase when expressed in E.coli, resulting in a mature sequence without the N-terminal methionine;the expected mature form of the PKE fusion proteins without theN-terminal methionine is shown in the list below.

A human serum albumin sequence contained in some of the fusion proteinswas HuSA(C34A). Its amino acid sequence is:

(SEQ ID NO: 321) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL.

Another human serum albumin sequence contained in some of the fusionproteins was HSA (C34A, des Leu-585). Its amino acid sequence is:

(SEQ ID NO: 322) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALG.

G4S×3 refers to the sequence GGGGS repeated 3 times, i.e.,GGGGSGGGGSGGGGS.

Exemplary fusion proteins contained more than one modified FGF-21polypeptide, e.g., Compounds 141-146.

The sequences of the fusion protein compounds are shown below, andfeatures of these fusion proteins are summarized in FIG. 40A-C.

Compound 101: PKE(2)-L1-FGF21 (Compound 2) (SEQ ID NO: 401)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 102: PKE(2)-L2-FGF21 (Compound 2) (SEQ ID NO: 402)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 103: PKE(2)-L3-FGF21 (Compound 2) (SEQ ID NO: 403)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPEEEEDEEEEDHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 104: PKE(2)-L4-FGF21 (Compound 2)(SEQ ID NO: 404)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 105: PKE(2)-L5-FGF21 (Compound 2)(SEQ ID NO: 405)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPGSHHHHHHHHGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 106: PKE(2)-L6-FGF21 (Compound 2)(SEQ ID NO: 406)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 107: PKE(2)-L7-FGF21 (Compound 2)(SEQ ID NO: 407)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 108: PKE(2)-L8-FGF21 (Compound 2)(SEQ ID NO: 408)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPGSGSGSGSGSGSGSGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 109: PKE(2)-L9-FGF21 (Compound 2)(SEQ ID NO: 409)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSTPPTPSPSTPPTPSPSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 110: PKE(2)-L10-FGF21 (Compound 2)(SEQ ID NO: 410)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPRGGEEKKKEKEKEEQEERETKTPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 111: PKE(2)-L11-FGF21 (Compound 2) (SEQ ID NO: 411)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPGGGGSGGGGSGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 112: PKE(2)-L12-FGF21 (Compound 2) (SEQ ID NO: 412)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSPEPPTPEPPSPEPPTPEPPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 113: PKE(2)-L13-FGF21 (Compound 2) (SEQ ID NO: 413)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSTPPTPSPSTPPTPSPSPSTPPTPSPSTPPTPSPSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 114: PKE(2)-L14-FGF21 (Compound 2) (SEQ ID NO: 414)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 115: PKE(2)-L15-FGF21 (Compound 2) (SEQ ID NO: 415)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSPEPPTPEPPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 116: PKE(2)-L16-FGF21 (Compound 2)(SEQ ID NO: 416)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSPEPPTPEPPSPEPPTPEPPSPEPPTPEPPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 117: PKE(2)-L17-FGF21 (Compound 2) (SEQ ID NO: 417)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPTPEPPSPEPPTPEPPSPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 118: PKE(2)-L18-FGF21 (Compound 2)(SEQ ID NO: 418)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSPEPGGGSPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 119: PKE(2)-L19-FGF21 (Compound 2)(SEQ ID NO: 419)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSPEPEEEDPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 120: PKE(2)-L20-FGF21 (Compound 2)(SEQ ID NO: 420)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSPEPPTPEPEEEDPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 121: PKE(2)-L21-FGF21 (Compound 2)(SEQ ID NO: 421)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPTPEPPSPEPPTPEPEEEDPSPEPPTPEPPSPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 122: PKE(2)-L22-FGF21 (Compound 2) (SEQ ID NO: 422)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPTPEPPSPEPPTPEPGGGGSPSPEPPTPEPPSPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 123: PKE(2)-L23-FGF21 (Compound 2) (SEQ ID NO: 423)GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTPPSPEPTPEPSPEPPTPEPSPEPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 124: HuSA(C34A)-L201-FGF21 (Compound 2) (SEQ ID NO: 424)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 125: HuSA(C34A)-L202-FGF21 (Compound 2) (SEQ ID NO: 425)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 126: HuSA(C34A)-L203-FGF21 (Compound 2) (SEQ ID NO: 426)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGETGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 127: HuSA(C34A)-L204-FGF21 (Compound 2) (SEQ ID NO: 427)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 128: HuSA(C34A)-L205-FGF21 (Compound 2) (SEQ ID NO: 428)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGETGSSGEGTHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 129: HuSA(C34A)-L206-FGF21 (Compound 2) (SEQ ID NO: 429)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 130: HuSA(C34A)-L207-FGF21 (Compound 2) (SEQ ID NO: 430)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGETGSSGEGTGSTGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 131: HuSA(C34A)-L208-FGF21 (Compound 2) (SEQ ID NO: 431)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 132: HuSA(C34A)-L209-FGF21 (Compound 2) (SEQ ID NO: 432)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGETGSSGEGTGSTGSGAGESHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 133: HuSA(C34A, des Leu-585)-L211-FGF21 (Compound 2)(SEQ ID NO: 434)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 134: HuSA(C34A, des Leu-585)-L207-FGF21 (Compound 2)(SEQ ID NO: 435)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGGETGSSGEGTGSTGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 135: HuSA(C34A, des Leu-585)-L211-FGF21 (Compound 1)(SEQ ID NO: 436)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASCompound 136: HuSA(C34A, des Leu-585)-L207-FGF21 (Compound 1)(SEQ ID NO: 437)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGGETGSSGEGTGSTGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASCompound 137: FGF21 (Compound 2)-L205-HuSA(C34A)- (SEQ ID NO: 440)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASGETGSSGEGTDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLCompound 138: FGF21 (Compound 2)-L209-HuSA(C34A)- (SEQ ID NO: 441)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASGETGSSGEGTGSTGSGAGESDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLCompound 139: FGF21 (Compound 2)-L210-HuSA(C34A)- (SEQ ID NO: 442)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASGETGSSGEGTGSTGSGAGESGTGESGEGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLCompound 140: FGF21 (Compound 1)-L209-HuSA(C34A)- (SEQ ID NO: 443)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASGETGSSGEGTGSTGSGAGESDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLCompound 141: FGF21 (Compound 2)-L209-HuSA(C34A)-G4Sx3-FGF21 (Compound 2) (SEQ ID NO: 446)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASGETGSSGEGTGSTGSGAGESDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 142: FGF21 (Compound 1)-L209-HuSA(C34A)-G4Sx3-FGF21 (Compound 1) (SEQ ID NO: 447)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASGETGSSGEGTGSTGSGAGESDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASCompound 143: FGF21 (Compound 2)-L209-HuSA(C34A, des Leu-585)-G4Sx3-FGF21 (Compound 2) (SEQ ID NO: 448)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASGETGSSGEGTGSTGSGAGESDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 144: FGF21 (Compound 1)-L209-HuSA(C34A, des Leu-585)-G4Sx3-FGF21 (Compound 1) (SEQ ID NO: 449)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASGETGSSGEGTGSTGSGAGESDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASCompound 145: FGF21 (Compound 2)-L210-HuSA(C34A)-G4Sx3-FGF21 (Compound 2) (SEQ ID NO: 450)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASGETGSSGEGTGSTGSGAGESGTGESGEGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQERSPSYASCompound 146: FGF21 (Compound 1)-L210-HuSA(C34A)-G4Sx3-FGF21 (Compound 1) (SEQ ID NO: 451)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASGETGSSGEGTGSTGSGAGESGTGESGEGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS Compound 147: PKE(1)-L1 -FGF21 (Compound 2) (SEQ ID NO: 452)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSY ASCompound 148: PKE(1)-L2-FGF21 (Compound 2) (SEQ ID NO: 453)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 149: PKE(1)-L3-FGF21 (Compound 2) (SEQ ID NO: 454)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQEEEEDEEEEDHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 150: PKE(1)-L4-FGF21 (Compound 2) (SEQ ID NO: 455)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 151: PKE(1)-L5-FGF21 (Compound 2) (SEQ ID NO: 456)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQGSHHHHHHHHGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 152: PKE(1)-L6-FGF21 (Compound 2)(SEQ ID NO: 457)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 153: PKE(1)-L7-FGF21 (Compound 2)(SEQ ID NO: 458)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 154: PKE(1)-L8-FGF21 (Compound 2)(SEQ ID NO: 459)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQGSGSGSGSGSGSGSGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 155: PKE(1)-L9-FGF21 (Compound 2)(SEQ ID NO: 460)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSTPPTPSPSTPPTPSPSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 156: PKE(1)-L10-FGF21 (Compound 2)(SEQ ID NO: 461)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQRGGEEKKKEKEKEEQEERETKTPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 157: PKE(1)-L11-FGF21 (Compound 2)(SEQ ID NO: 462)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQGGGGSGGGGSGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 158: PKE(1)-L12-FGF21 (Compound 2)(SEQ ID NO: 463)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSPEPPTPEPPSPEPPTPEPPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 159: PKE(1)-L13-FGF21 (Compound 2)(SEQ ID NO: 464)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSTPPTPSPSTPPTPSPSPSTPPTPSPSTPPTPSPSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 160: PKE(1)-L14-FGF21 (Compound 2) (SEQ ID NO: 465)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 161: PKE(1)-L15-FGF21 (Compound 2) (SEQ ID NO: 466)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSPEPPTPEPPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 162: PKE(1)-L16-FGF21 (Compound 2)(SEQ ID NO: 467)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSPEPPTPEPPSPEPPTPEPPSPEPPTPEPPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 163: PKE(1)-L17-FGF21 (Compound 2) (SEQ ID NO: 468)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPTPEPPSPEPPTPEPPSPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 164: PKE(1)-L18-FGF21 (Compound 2)(SEQ ID NO: 469)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSPEPGGGSPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 165: PKE(1)-L19-FGF21 (Compound 2)(SEQ ID NO: 470)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSPEPEEEDPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 166: PKE(1)-L20-FGF21 (Compound 2)(SEQ ID NO: 471)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSPEPPTPEPEEEDPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 167: PKE(1)-L21-FGF21 (Compound 2)(SEQ ID NO: 472)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPTPEPPSPEPPTPEPEEEDPSPEPPTPEPPSPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 168: PKE(1)-L22-FGF21 (Compound 2) (SEQ ID NO: 473)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPTPEPPSPEPPTPEPGGGGSPSPEPPTPEPPSPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 169: PKE(1)-L23-FGF21 (Compound 2) (SEQ ID NO: 474)GVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEKPSQPSPEPTPEPSPEPPTPEPSPEPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 170 [Fc(hIgG1a_191)-L7-FGF21(Cmp. 2)](SEQ ID NO: 475)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 171 [Fc(hIgG1a_191)- L250-FGF21(Cmp. 2)] (SEQ ID NO: 476)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGGSGGGSGGGGSGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 172 [Fc(hIgG1a_191)-L12-FGF21(Cmp. 2)] (SEQ ID NO: 477)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPSPEPPTPEPPSPEPPTPEPPSPEPPTPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 173 [Fc(hIgG1a_191)-L251-FGF21(Cmp. 2)] (SEQ ID NO: 478)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPSPEPPTPEPPSPEPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 174 [Fc(hIgG1a_191)-L5-FGF21(Cmp. 2)](SEQ ID NO: 479)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSHHHHHHHHGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 175 [Fc(hIgG1a_191)-L252-FGF21(Cmp. 2)](SEQ ID NO: 480)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKELQLEESAAEAQEGELEHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 176 [Fc(hIgG1a_190)-L253-FGF21(Cmp. 2)] (SEQ ID NO: 481)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSSGGGGSGGGSGGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 177 [Fc(hIgG1a_191)-L6-FGF21(Cmp. 2)] (SEQ ID NO: 482)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS Compound 178 [Fc(hIgG1a_189)-L6-FGF21(Cmp. 2)](SEQ ID NO: 483)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 179 [Fc(hIgGla_191)-L7-FGF21-1aa(Cmp. 2 with the C-terminalamino acid deleted)] (SEQ ID NO: 484)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYACompound 180 [Fc(hIgG1a_191)-L7-FGF21-3aa(Cmp.2 with the three C-terminalamino acids deleted)] (SEQ ID NO: 485)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSCompound 181 [Fc(hIgG1f1.1_186)-L7-FGF21 (Cmp. 2)] (SEQ ID NO: 486)EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYASCompound 182 [Fc(hIgG1a_191b)-L7-FGF21(Cmp. 2)] (SEQ ID NO: 487)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGSGGGSGGGGSHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLGSGRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS

Example 5 In Vitro Potency of FGF-21 Deletion Molecules

In this example, the potency of several modified FGF-21 polypeptides istested and compared to the potency of Pegylated Compound 1 in acell-based in vitro receptor activation assay. It is shown that severalmodified FGF-21 polypeptides, including Pegylated Compound 2 havecomparable potency to Pegylated Compound 1.

Methods

A clonal human embryonic kidney (HEK) 293 cell line stably expressinghuman β-klotho was generated to characterize FGF21 variants in theprimary in vitro assay. HEK-293 cells were transfected by following themanufacturer's protocol for the Lipofectamine 2000 (Invitrogen)transfection reagent and using a linearized plasmid encoding humanβ-klotho with a C-terminal FLAG tag (N-DYKDDDDK-C in one letter aminoacid code) under the control of a cytomegalovirus promoter. Positiveclones were isolated after 14 days of growth in selection medium [600μg/mL of Geneticin (Invitrogen) in Dulbecco's modified Eagle's medium(DMEM) with 4.5 g/l D-glucose containing L-Glutamine, Hepes (Invitrogen)and 10% fetal bovine serum (FBS)].

The primary assay measured FGF21 variant-dependent phosphorylation ofextracellular signal-regulated kinase 1/2 (pERK1/2) in a clonal HEK-293cell line stably expressing human β-klotho. For assays, the cells wereseeded into 96-well tissue culture plates (Falcon) at 40,000 cells perwell in 200 μL of selection medium and maintained at 37° C. in ahumidified 5% CO₂ atmosphere. After 48 hours, the full selection mediumwas replaced with serum-free medium (DMEM with 4.5 g/l D-glucose and0.1% fatty acid free bovine serum albumin), and the cells weremaintained for 6 hours at 37° C. in a humidified 5% CO₂ atmosphere. Thecompounds to be tested were serially diluted in serum-free medium. Theassay was initiated by replacing the serum-free medium on the cells withthe diluted compounds, the incubation was allowed to proceed for 7minutes at room temperature, and the reaction was terminated by removingthe compounds and adding 100 μL of lysis buffer (Perkin ElmerAlphaScreen kit) to each well. The plates were shaken at ˜80 rpm forapproximately 10 minutes at room temperature and stored frozen at −80°C. An aliquot (4 μl) from each well of the thawed cell lysate wasanalyzed for pERK1/2 according to the manufacturer's protocol for theSurefire AlphaScreen pERK1/2 kit and the AlphaScreen IgG Detection kit,protein A, (Perkin Elmer) using 384 well white Proxiplates (PerkinElmer). Plates were incubated at room temperature for two hours in thedark and then read on an Envision 2103 Multiplate reader (Perkin Elmer)using the AlphaScreen protocol. Data were fit to a 4 parameterlog(agonist) vs. response equation by non-linear regression usingGraphPad Prism 5 software.

Results

In vitro potency was determined by measuring FGF21-dependentphosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in ahuman embryonic kidney (HEK) 293 cell line in which human β-Klotho hasbeen stably introduced to function as a co-receptor with endogenouslyexpressed FGFR splice variants. Potency was evaluated by determining themean EC₅₀ or pEC₅₀ (negative base 10 log of the EC₅₀ concentrationexpressed in moles per liter) value for each tested compound. Anexemplary dose-response curve is shown in FIG. 2 for Pegylated Compound1 and Pegylated Compound 2. Results for additional tested compounds areshown in Tables 4 and 5.

TABLE 4 EC50 values for in vitro modified FGF-21 activity in the pERKassay. pERK EC50 Compound Name (nM) n = 3 Pegylated Compound 1 6.7Compound 2 4.6 Compound 3 28 Compound 5 69.2 Compound 6 39.1 Compound 728.4 Compound 10 21.9 Compound 11 28.2 Compound 12 31.9 Compound 19 5.1Compound 20 3.9 Compound 22 4.1 Compound 23 30

TABLE 5 EC50 values for in vitro modified FGF-21 activity in the pERKassay. Compound Name EC50 (nM) Pegylated Compound 1 14 PegylatedCompound 2 28 Pegylated Compound 5 24 Pegylated Compound 6 20 PegylatedCompound 10 28 Pegylated Compound 11 30 Pegylated Compound 12 39Pegylated Compound 19 30 Pegylated Compound 20 42 Pegylated Compound 2130 Pegylated Compound 22 36 Compound 32 8 (Non-Pegylated Control)

Example 6 Thermal Stability Testing of Modified FGF-21 Polypeptides

In this example, thermal stability of modified FGF-21 polypeptides wasmeasured using Thermal Scanning Fluorescence (TSF) and DifferentialScanning calorimetry (DSC). Thermal stability is recognized in theliterature to have predictive value for determining propensity to formaggregates (see, e.g., Webster, “Predicting Long-Term Storage Stabilityof Therapeutic Proteins,” Pharmaceutical Technology, Volume 37, Issue11, pp. 42-48, Nov. 2, 2013).

Methods

Thermal midpoints of denaturation were measured by differential scanningcalorimetry in a MicroCal (Malvern Instruments) auto VP-DSC. For DSCanalysis, the protein samples were formulated in 250 mM sucrose, 20 mmhistidine pH 7.0 at approximately 2 mg/mL with a scan rate of 90° C. perhour. The reference cell was filled with the identical buffer sansprotein. The transition midpoints (Tm) of the phase change betweenfolded and thermally unfolded protein domains under the solutionconditions and scan rate applied was determined from the peak maximumsseen in the thermogram trace (arrows).

For thermal scanning fluorescence (TSF), protein is diluted to 0.2 mg/mLinto the buffer of interest and a small amount of fluorescentfluorophore anilinonaphthalene sulfonic acid (ANS) is added. Samples areplaced in a 96 well PCR thin wall plate and the temperature of theprotein sample in the presence of the extrinsic fluorophore is increasedwhile the fluorescence of the sample is monitored on a Bio-Rad CT1000-RTPCR instrument. The midpoint of protein thermal unfolding of the proteinis determined by monitoring the increase in fluorescent signal of theprobe as it interacts with the newly exposed hydrophobic core ofthermally denaturing protein. The temperature at the midpoint ofunfolding (Tm) under the buffer and scan rate conditions examined wasdefined as the temperature inflection point of the fluorescent signalrise curve.

Results

Representative DSC scanning results are shown in FIG. 3. PegylatedCompound 2 was observed to have a transition midpoint (“Tm”) temperatureapproximately 8 degrees C. higher than Pegylated Compound 1. Thermalreversibility was >95% (data not shown).

FIG. 4 shows representative results for TSF. Compound 10 exhibited anapproximately 8 degrees C. increase in Tm relative to Compound 1 (havinga wild-type FGF-21 sequence except that an N-terminal methionineincluded for expression in E. coli). Results for additional compoundsare shown in Table 6, below.

Together these results indicate that exemplary compounds would bereasonably expected to have a decreased propensity to form aggregates,for example, Compounds 2, 10, and 16. The decreased propensity to formaggregates is expected to confer a longer shelf-life and/or the abilityto be formulated to a greater concentration.

TABLE 6 Thermal stability results determined by thermal scanningfluorescence for modified FGF-21 polypeptides compared to Compound 1.Increased thermal stability was observed up to approximately 7-8 degreesC. for some compounds. Tm Compound (degrees C.) Difference from Compound1 Compound 1 46.63 0 Compound 2 53.2 6.57 Compound 3 46.92 0.29 Compound5 47.46 0.83 Compound 6 43.2 −3.43 Compound 7 42.78 −3.85 Compound 1054.5 7.87 Compound 11 43.9 −2.73 Compound 12 45.4 −1.23 Compound 1442.97 −3.66 Compound 16 50.8 4.17 Compound 18 47.96 1.33

Example 7 Deamidation and Aggregate Formation

This example describes assessment of deamidation and aggregate formationfor of modified FGF-21 polypeptides.

Methods

Thermal Stress Tests:

Protein samples of interest are prepared in the test buffer (250 mMsucrose, 20 mM Histidine, pH 6.5, and pH 7 or 20 mM TRIS pH 7.5) at aconcentration of 7.5 mg protein/mL. Samples were examined by analyticalsize exclusion chromatography (aSEC) and charge variant analysis byimaged capillary isoelectric focusing (icIEF) at time=0. Samples werethen stress tested by placing them in an incubator 25° C. for 1 week andthen at 40° C. for 5 weeks. Aliquots were withdrawn and examined byicIEF and aSEC at various time pointS over the next 4 to 5 weeks werewithdrawn and further examined by aSEC and icIEF to monitor stability.

aSEC

The size exclusion chromatography (aSEC) was conducted using Agilent1100 HPLC system (Agilent Technologies, Santa Clara, Calif. USA) on aZenix-C 300 SEC column (Sepax Technologies, Newark, Del. USA) with adimension of 300×4.6 mm. The mobile phase used was 200 mM potassiumphosphate and 150 mM sodium chloride adjusted to pH 6.9, at a flow rateof 0.35 mL/min. Approximately 20 μg sample was injected for eachanalysis. The separation was monitored at UV 280 nm. Quantification ofmonomer vs HMW species was performed by integration of the area underthe curve at retention times corresponding to each species.

icIEF

Deamidation was detected by charge variant analysis (deamidationincreases the net negative charge of the protein and the formation ofacidic variants) conducted by imaged capillary isoelectric focusing(icIEF), which was performed on an iCE280 instrument (ProteinSimple,Santa Clara, Calif. USA) using a fluorocarbon coated capillary, 100μm×50 mm. The sample was diluted to 0.3 mg/mL using a solution thatcontains 0.35% methyl cellulose, 4% pH3-10 Pharmalytes, 4M urea, and 2%pI marker. The anolyte and catholyte are 0.08 M phosphoric acid and 0.1M sodium hydroxide, respectively. The focusing was achieved by applying1.5 kV for 2 min and then 3.0 kV for 11 min. Quantification of (−)charge modified species relative to the parent species was performed byintegration of the corresponding area under the curves.

Results

The biophysical properties of Pegylated Compound 2 demonstratedsuperiority over Pegylated Compound 1. No degradation was observedthrough 5 weeks at 2-8° C. coupled with low rates of degradationobserved under accelerated in conditions at 40° C. relative to PegylatedCompound 1 (FIG. 5). Eight week accelerated studies indicate thatPegylated Compound 2 is superior to Pegylated Compound 1 in the testedformulation (Histidine/Sucrose pH7.0). Under these excipient conditions,deamidation is abrogated and Pegylated Compound 2 has a decreasedpropensity to form soluble high molecular weight (HMW) aggregates. Theseresults suggest Pegylated Compound 2 has a broader pH window forformulation options with lessened aggregation propensities relative toPegylated Compound 1.

HMW aggregate formation was also assessed. Lower HMW aggregate formationat a given concentration is indicative of greater solubility, whichwould result in the ability to be formulated to a higher concentration.FIG. 6 illustrates HMW aggregate formation for several modified FGF-21polypeptides and Compound 1. For the majority of tested compounds,aggregate formation was decreased relative to Pegylated Compound 1 (e.g.Pegylated Compound 2 and Pegylated Compound 10). For a few compounds,HMW aggregate formation was similar to or higher than for PegylatedCompound 1.

Deamidation was also assessed. FIG. 7 illustrates the detected levels ofdeamidation (indicated by formation of acidic variants over time) forseveral modified FGF-21 polypeptides and Pegylated Compound 1. For allmodified FGF-21 polypeptides shown, the level of deamidation detectedwas decreased relative to Pegylated Compound 1.

Example 8 Solubility Assessment of Modified FGF-21 Polypeptides

This example describes measurement of the relative solubility ofmodified FGF-21 polypeptides. The results are indicative of the abilityof a compound to be formulated to a relatively higher concentration,which would permit more facile administration of an effective dosage.

Methods

Relative solubility assessments were performed by sequential plug-flowconcentration cycles followed by size-exclusion chromatography analysis.Samples, formulated in PBS pH 7.2 at similar but not identical startingconcentrations, were pipetted into 3 kDa molecular weight cut-offcentrifuge concentrators and spun at 4,750 RPM for 10 minute, 15 minute,and 30 minute cycles at 4° C. In between spin cycles, aliquots wereremoved from the concentration apparatus and analytical size exclusionchromatography analysis (aSEC) was performed (on a GE HealthcareSuperdex S-75 10/300 GL column equilibrated in PBS pH 7.2 buffer) todetermine the concentration of HMW and monomer species in the solution.

Total Concentrations were determined by absorbance at 280 nm with aNanoDrop spectrophotometer. High molecular weight percentage wasdetermined by area under the curve calculations of high molecular weightpeaks relative to the monomer peaks in the SEC chromatogram trace. Theresulting data points are plotted to visualize the rank order of leastsoluble constructs to most soluble under the conditions tested, thelower the slope created by the data points the more stable the proteinvariant under the conditions tested

Results

Relative solubility of Modified FGF-21 polypeptides was determined bymeasuring the formation of high molecular weight (HMW) aggregates as afunction of protein concentration. Lower HMW aggregate formation at agiven concentration is indicative of greater solubility, which wouldresult in the ability to be formulated to a higher concentration.

The slope of the plug flow solubility curve for the tested compounds isshown below in Table 7 (lower values indicate less aggregate formationand hence the ability to be formulated to a higher concentration).Relatively low aggregate formation levels were observed for several ofthe compounds (e.g. Compound 2 and Compound 10), indicating loweraggregate formation and greater solubility (FIG. 8 and Table 7). Thehighest plug flow solubility slope was observed for Compound 1.

TABLE 7 Plug Flow Solubility Results for modified FGF-21 Polypeptides.Plug Flow Solubility Compound Slope Compound 1 1.863 Compound 2 0.3171Compound 3 1.2219 Compound 5 0.8548 Compound 6 1.1353 Compound 7 1.4318Compound 10 0.3647 Compound 11 1.1156 Compound 12 1.0317 Compound 190.9743 Compound 20 1.1545 Compound 22 0.1997 Compound 23 0.771

Example 9 Immunogenicity Testing of FGF-21 Deletion Molecules

T-cell activation response studies were performed, and indicate theprotein sequence used in the majority of the compounds presented anequivalent response to the protein sequence used in Pegylated Compound1.

Methods

Immunogenicity was assessed by a CD4+ T cell proliferation assay.Peripheral blood mononuclear cells (PBMCs) from a diverse set of humanblood donors were isolated using a Ficoll gradient. Donors were HLAtyped to ensure coverage of the variability in the MHC class II allelepresent in the human population. After labeling, the PBMCs withCarboxyfluorescein succinimidyl ester (CFSE) were plated in 96 wellformat at 200,000 cells per well in standard cell culture media in thepresence of the tested compound for 7 days. Proliferation of the CD4+ Tcells was analyzed by labeling with an anti CD4 antibody and flowcytometry. The percentage antigenicity protein's is calculated as thepercentage of donors that show significant CD4+ T cell proliferationresponse vs a media control.

Results

In an in vitro immunogenicity risk assessment utilizing a human T cellproliferation assay 13 out of 40 donors (32.5%) showed CD4+proliferative response after 7 days of exposure to Compound 2 comparedto 15 out of 40 donors that showed a positive signal from Compound 1(having a wild-type FGF-21 sequence except that an N-terminal methionineincluded for expression in E. coli) (FIG. 9). While this cell basedexperiment does not replace real world human immunlogic responseobservations this analysis suggests that there is no increased humanimmunogenicity risk for Compound 2 compared to wild type FGF21.

Further modified FGF-21 polypeptides were tested in the CD4+proliferation assay relative to Compound 1 (FIG. 9) Immunogenicity wasgenerally similar to that observed for the control FGF21 sequence(Compound 1), with the exception of Compound 10 which exhibited arelatively higher immune response, indicating that the compound might beundesirably immunogenic if administered to human patients.

Example 10 In Vivo Stability of C-Terminally Intact (Active) ModifiedFGF-21 Polypeptides

This study assessed the level of the C-terminally intact (i.e., active)modified FGF-21 polypeptides in vivo. Pegylated Compound 2 exhibited agreatly increased proportion of C-terminally intact, active polypeptidecompared with Pegylated Compound 1, including a greater duration of invivo activity.

Methods

The pharmacokinetics of Pegylated Compound 2 and Pegylated Compound 1were evaluated in Cynomolgus monkeys following a subcutaneous (SC) doseof 0.25 mg/Kg and 0.225 mg/kg respectively. Blood samples (0.2 mL) werecollected at 0, 0.25, 0.5, 1, 3, 7, 24, 48, 72, 96, 120, 144, 168 hrfollowing drug administration and stored at −80° C. until bioanalysis.

The PK parameters of total and C-terminal intact Pegylated Compound 1and Pegylated Compound 2 were obtained by non-compartmental analysis ofserum or plasma concentration vs time data (Phoenix™ WinNonlin®, 6.3,Pharsight Corporation, St. Louis, Mo.). The area under the curve fromtime zero to infinity [AUC(tot)] were calculated using a combination oflinear and log trapezoidal summations. Estimations of AUC and half-life(t ½) were made using a minimum of 3 timepoints with quantifiableconcentrations.

Concentrations of total Pegylated Compound 1 in the single-dose PKstudies were measured using a non-validated, Meso Scale Discovery(MSD)-based electrochemiluminescent immunosorbent assay (ECLIA). APEG-specific monoclonal antibody (mAb) was used to capture PegylatedCompound 1, followed by the use of a rabbit anti-FGF21 polyclonalantibody (pAb) for detection of total Pegylated Compound 1.

The subcutaneous (SC) pharmacokinetics (PK) of Pegylated Compound 2 andPegylated Compound 1 were also evaluated in male Ob/Ob mice. PegylatedCompound 2 was administered to mice (n=3/timepoint/route) as a single SC0.1 mg/kg injection. Pegylated Compound 1 was administered as a singleSC dose of 0.05 mg/kg. Blood samples were collected at various timepoints following drug administration and processed essentially asdescribed for the cynomolgus monkeys.

The read-out was via electrochemiluminescence, expressed in relativelight units (RLU), which was proportional to the amount of totalPegylated Compound 1 bound by the capture and detection reagents. Thestandard curves were between 0.2 and 154 ng/mL. Test samples werequantified using a 4-parameter logistic fit regression model with aweighting factor of 1/Y. The same assay was used to measure theconcentrations of C-terminal intact Pegylated Compound 1 in ZDF rat andcynomolgus monkey serum except a rabbit anti-FGF21 pAb specific to theC-terminus of Pegylated Compound 1 was used for detection. The standardcurves were between 0.2 and 154 ng/mL. Test samples were quantifiedusing a 4-parameter logistic fit regression model with a weightingfactor of 1/Y.

Results

Pegylated Compound 1 undergoes proteolysis in vivo, such that over timethe majority of the compound becomes a truncated, inactive form. The invivo proteolytic stability of Pegylated Compounds 1 and 2 was comparedhead-to-head in ob/ob mice (FIG. 10) and cynomolgus monkeys (FIG. 11).These results show that the AUC of the active C-terminal intact formincreased by 7- to 8-fold for the modified FGF-21 compound containingthe deletion (Pegylated Compound 2) as compared with Pegylated Compound1, indicating Pegylated Compound 2 has increased in vivo proteolyticstability.

Example 11 Pharmacokinetic Studies of Modified FGF-21 Polypeptides

This example presents the results of pharmacokinetic studies usingmodified FGF-21 polypeptides in mice, rats, and cynomolgus monkeys (seeTable 8, below).

Briefly, the clearance of Pegylated Compound 2 was low in mice, rats,and monkeys (range: 0.94-2.6 mL/h/kg). The volume of distribution (Vss),at ˜0.04-0.05 L/kg, was close to plasma volume and indicated minimaldistribution into the extravascular space. The subcutaneous (SC)bioavailability was acceptable in mice (40%) and monkeys (58%), while itwas relatively low in rats (16%). This low SC bioavailability could berat-specific, as other PEGylated molecules have been observed to have asimilar phenomenon (low SC bioavailability in rats with goodbioavailability in mice, monkeys, and humans). More importantly, the SCbioavailability for Pegylated Compound 2 in these species (including therat) was 2-3.4 fold higher relative to Pegylated Compound 1.

Following SC administration of Pegylated Compound 2 to mice, rats, andmonkeys, there was a consistent (7-8 fold) increase in dose normalizedexposure (AUC) of intact protein, relative to Pegylated Compound 1. Theincreased exposures of intact Pegylated Compound 2 after subcutaneousadministration can be attributed to lower systemic clearance (2-5 fold)along with an improvement (2-3.4 fold) in subcutaneous bioavailabilityin all the three species relative to Pegylated Compound 1.

Total Pegylated Compound 2, composed of a mixture of proteolyticfragments and intact Pegylated Compound 2, was also measured in thesepharmacokinetic studies. The AUC ratio of intact Pegylated Compound 2over total Pegylated Compound 2 in mice and rats was close to 1following IV administration indicating minimal systemic proteolysis.However, following SC administration, the ratio was 0.6-0.86 in thesespecies indicating some proteolysis in the SC site. A similar trend wasnoted in monkeys wherein the AUC ratio of intact Pegylated Compound 2over total Pegylated Compound 2 was modestly lower after SCadministration compared to IV (0.7 after SC vs. 0.8 after IV dosing).

Overall, Pegylated Compound 2 demonstrated favorable in vitro and invivo pharmacokinetic properties including increased bioavailability,desceased clearance and increased AUC (area under the plasmaconcentration-time curve).

TABLE 8 IV & SC Pharmacokinetics of Intact modified FGF-21 Protein inMouse, Rat, and Monkey. Species Mouse Rat Monkey Pegylated PegylatedPegylated Pegylated Pegylated Pegylated Compound 1 Compound 2 Compound 1Compound 2 Compound 1 Compound 2 CL 6.1 2.5 6.2 ± 0.7 2.6 ± 0.4 4.4 ±1   0.94 ± 0.05 (mL/h/kg) Vss (L/kg) 0.043 0.044 0.05 ± 0.01 0.05 ± 0.01 0.07 ± 0.005  0.04 ± 0.003 Half Life (h) 47 26 35 ± 1   24 ± 3.9 13 ±2   57 ± 4.2 MRT (h) 7.1 20 8.4 ± 0.3  20 ± 1.3  16 ± 2.5  47 ± 1.5 SCBA (%) 15 40 4.8 16.3 30 58 SC AUC 1.2 8.1 0.39 ± 0.07 3.1 ± 0.2  20 ±2.2 154 ± 63  (μg/mL * h) Abbreviations: CL: Apparent total bodyclearance of the drug from plasma; Vss: Apparent volume of distributionat steady state; MRT: Mean residence time; SC BA: subcutaneousbioavailability; SC AUC: subcutaneous area under the plasmaconcentration-time curve.

Example 12 Repeated Dosing In Vivo Studies of Modified FGF-21Polypeptides in a Mouse Model of Diabetes

Pegylated Compound 2 and Pegylated Compound 1 were evaluatedhead-to-head in a 21-day repeated dosing study. The results demonstratethat while both compounds were effective for ameliorating metabolicsymptoms, the longer in vivo half-life of the active Pegylated Compound2 resulted in greater therapeutic effects, particularly when comparingthe effects of weekly dosing.

Methods

Male ob/ob mice (Jackson Laboratories, Bar Harbor, Me.) were 8 weeks ofage at the start of the study. Mice were randomized into treatmentgroups based on body weight and glucose levels. All groups were treatedby subcutaneous (s.c.) administration of 1 ml/kg dosing solutions. Thetreatment groups were as follows: 1) vehicle (250 mM sucrose/20 mM Tris,pH 8.3) twice weekly (BIW), 2) Pegylated Compound 1, 0.15 mg/kg BIW, 3)Pegylated Compound 2, 0.15 mg/kg BIW, 4) Pegylated Compound 1, 0.3 mg/kgonce weekly (QW), or 5) Pegylated Compound 2, 0.3 mg/kg QW. Mice thatwere only administered compound once weekly were administered vehicle onthe days when BIW groups were administered their second weekly injectionof compound. Body weight, plasma glucose, triglycerides (Olympusclinical chemistry analyzer, AU680), and insulin (ELISA, Mercodia Inc.)were determined throughout the 21 day dosing period in the fed state.Glycated hemoglobin (HbA1c) was determined in whole blood at study startand termination, also with the Olympus analyzer.

Results

Both Pegylated Compound 1 and Pegylated Compound 2 were administeredsubcutaneously using doses of 0.15 mg/kg twice weekly (BIW) and 0.3mg/kg QW. The plasma concentrations measured at trough on day 21 showthat exposure of total (intact and proteolyzed) FGF21 was similar forboth variants, but exposure of active, C-terminal intact PegylatedCompound 2 was 12- to 25-fold higher than that of Pegylated Compound 1following QW or BIW administration, respectively (Table 9).

TABLE 9 Exposure of C-terminal intact Pegylated Compound 1 and PegylatedCompound 2 at trough after 21 days of repeated dosing in ob/ob mice(mean ± s.e.m.) Pegylated Pegylated Pegylated Pegylated CompoundCompound Compound Compound 2 BIW 1 BIW 2 QW 1 QW Parameter (0.15 mg/kg)(0.15 mg/kg) (0.3 mg/kg) (0.3 mg/kg) Conc. at trough 175 ± 43 7 ± 0.7 38± 3 3 ± 0.1 (ng/mL) Projected 1064 313 2149 633 Cmax (ng/mL)* ProjectedAUC 107 18 107 18 (μg/mL * h)* *Projected based on single dosepharmacokinetic data in ob/ob mice

Glycated hemoglobin (HbA1c) is generated in vivo from the nonenzymaticaddition of glucose to specific amino acids within this protein, and thepercent HbA1c measured corresponds to the average blood glucoseintegrated over the lifetime of circulating erythrocytes. An HbA1c valuegreater than 6.5% is a diagnostic criterion for diabetes. Thevehicle-corrected changes in HbA1c on day 21 of the repeated dosingstudy are shown in FIG. 12. BIW administration of 0.15 mg/kg of eitherFGF21 polypeptides normalized HbA1c to values less than 5% following 21days of administration. However, only Pegylated Compound 2 statisticallysignificantly reduced ΔHbA1c vs. vehicle with QW administration of 0.3mg/kg, consistent with the significant increase in functional exposureto active C-terminal intact protein. These results indicate thatmaintenance of a target trough concentration (C_(trough)) can besufficient for efficacy (Table 9).

Consistent with the decreased HbA1c results, plasma glucose levels werealso significantly decreased throughout the study (all groups p<0.01 fordays 2 to 21 versus vehicle except the QW doses on day 14, for which thePegylated Compound 2 achieved statistical significance (p<0.05) andPegylated Compound 1 was not significant) (FIG. 16). Overall, the changein glucose AUC (percentage difference from vehicle) was significantlydecreased by all treatments over the course of the study (p<0.001 vsvehicle) (FIG. 17).

Additionally, plasma triglyceride levels were significantly reduced inthe Pegylated Compound 2 group vs. vehicle, on days 2 and 4 for the BIWgroup (p<0.01) and on days 2, 4 and 10 and 17 for the QW group (p<0.01),as well as on days 2 and 17 for the Pegylated Compound 1 QW group(p<0.05) (FIG. 18).

Pegylated Compound 2 significantly reduced percent body weight gainrelative to vehicle or Pegylated Compound 1 throughout the course of the21-day study in ob/ob mice (FIG. 13 and FIG. 14). QW administration of0.3 mg/kg Pegylated Compound 2 shows reduced efficacy on days 7, 14, and21 when body weight was measured at trough, again indicating thatmaintenance of a target trough concentration (C_(trough)) can besufficient for efficacy.

BIW administration of 0.15 mg/kg Pegylated Compound 2 statisticallysignificantly reduced plasma insulin levels throughout the 21-dayrepeated dosing study in ob/ob mice (FIG. 15); BIW Pegylated Compound 1failed to significantly reduce plasma insulin after Day 10. QWadministration of 0.3 mg/kg Pegylated Compound 2 gave rise to anapparent “saw-toothed” profile with a trend toward exceeding the valuesin the vehicle group on Days 14 and 21 when insulin was measured atcompound trough. This pattern suggests that Pegylated Compound 2 maypreserve insulin content in the pancreas with chronic dosing.

These results show that maintenance of a trough concentration of 175ng/ml (Table 9) for Pegylated Compound 2 in the 0.15 mg/kg BIW armresulted in significant improvements in measures of glycemia and bodyweight changes.

Relative to Pegylated Compound 1, the increased exposure to activeC-terminal intact Pegylated Compound 2 in the ob/ob mouse model resultedin enhanced in vivo potency on a dose-basis and extended duration ofaction with QW administration leading to superior reductions in HbA1c,weight gain, and plasma insulin.

Example 13

Progression from fatty liver to nonalcoholic steatohepatitis (NASH) canultimately result in liver fibrosis. This example describes the use ofan FGF-21 polypeptide comprising a human FGF-21 polypeptide modified tocontain a substitution of para-acetyl-L-phenylalanine for glutamine atposition 108 and linked to a 30 kDa poly(ethylene glycol) (PegylatedCompound 1 or “PEG-Compound 1,” SEQ ID NO:201) in a model of fibrosis.Specifically, PEG-Compound 1 was tested in the Stelic Institute's 2-hitmodel of NASH, in which C57BL6 mice are treated with streptozotocinshortly after birth to induce diabetes, and then placed on a high fatdiet at 4 weeks of age. These mice develop fatty liver (5-weeks), NASH(7-weeks), liver fibrosis (9-weeks), and ultimately hepatocellularcarcinoma (16 weeks) over a reproducible time-course. PEG-Compound 1prevented or effectively reversed the development of NASH in the Stelicmodel when the mice were treated from 5- to 9-weeks of age (preventativemodel) or 7- to 9-weeks of age (therapeutic model). PEG-Compound 1 alsodecreased liver fibrosis at the 9-week time point in this study.

The sequence of PEG-Compound 1 is as follows:

(SEQ ID NO: 201) MHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVY(pAF)SEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS, where pAF islinked to a 30 kDa PEG.

Materials and Methods

C57BL/6 mice (15-day-pregnant females) were obtained from Charles RiverLaboratories Japan, Inc. (Kanagawa, Japan). All animals used in thestudy were housed and cared for in accordance with the JapanesePharmacological Society Guidelines for Animal Use.

The animals were maintained in a SPF facility under controlledconditions of temperature (23±2° C.), humidity (45±10%), lighting(12-hour artificial light and dark cycles; light from 8:00 to 20:00) andair exchange. A high pressure (20±4 Pa) was maintained in theexperimental room to prevent contamination of the facility. The animalswere housed in polycarbonate cages KN-600 (Natsume Seisakusho, Japan)with a maximum of 4 mice per cage. Sterilized PULMASμ (Material ResearchCenter, Japan) was used for bedding and replaced once a week.

Sterilized solid high fat diet (HFD) was provided ad libitum, beingplaced in the metal lid on top of the cage. Pure water was provided adlibitum from a water bottle equipped with a rubber stopper and a sippertube. Water bottles were replaced once a week, cleaned and sterilized inautoclave and reused.

NASH was induced in 40 male mice by a single subcutaneous injection of200 μg streptozotocin (STZ, Sigma-Aldrich Co. LLC., USA) solution 2 daysafter birth and feeding with high fat diet (HFD, 57 kcal % fat, cat#:HFD32, CLEA Japan, Inc., Japan) after 4 weeks of age (“STAM mice”). Foodconsumption was measured daily per week during the treatment period.

PEG-Compound 1 and vehicle (20 mM Tris/250 mM sucrose, pH 8.3) wereadministered by subcutaneous route in a volume of 1 mL/kg. PEG-Compound1 was administered at doses of 1 and 3 mg/kg twice per week.

Non-fasting blood glucose was measured in whole blood using LIFE CHECK(EIDIA Co. Ltd., Japan). For plasma biochemistry, blood was collected inpolypropylene tubes with anticoagulant (Novo-Heparin, MochidaPharmaceutical Co. Ltd., Japan) and centrifuged at 1,000×g for 15minutes at 4° C. The supernatant was collected and stored at −80° C.until use. Plasma Alanine aminotransferase (ALT), triglyceride and totalcholesterol levels were measured by FUJI DRI-CHEM 7000 (FujifilmCorporation, Japan).

Liver total lipid-extracts were obtained by Folch's method (Folch J. etal., J. Biol. Chem. 1957; 226: 497). Liver samples were homogenized inchloroform-methanol (2:1, v/v) and incubated overnight at roomtemperature. After washing with chloroform-methanol-water (8:4:3,v/v/v), the extracts were evaporated to dryness, and dissolved inisopropanol. Liver triglyceride and cholesterol contents were measuredby Triglyceride E-test and Cholesterol E-test, respectively (Wako PureChemical Industries, Ltd., Japan).

For hematoxylin and eosin (HE) staining, sections were cut from paraffinblocks of liver tissue prefixed in Bouin's solution and stained withLillie-Mayer's Hematoxylin (Muto Pure Chemicals Co., Ltd., Japan) andeosin solution (Wako Pure Chemical Industries). Nonalcoholic fatty liverdisease (NAFLD) Activity score (NAS) was calculated according to thecriteria of Kleiner (Kleiner D E. et al., Hepatology, 2005; 41:1313). Tovisualize macro- and microvesicular fat, cryosections were cut fromfrozen liver tissues, prefixed in 10% neutral buffered formalin,embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Japan Co. Ltd.,Japan), and stained with Oil Red O (Sigma-Aldrich). Forimmunohistochemistry, sections were cut from frozen liver tissuesembedded in Tissue-Tek O.C.T. compound and fixed in acetone. Endogenousperoxidase activity was blocked using 0.03% H2O2 for 5 minutes, followedby incubation with Block Ace (Dainippon Sumitomo Pharma Co. Ltd., Japan)for 10 minutes. The sections were incubated with a 200-fold dilution ofanti-F4/80 antibody (BMA Biomedicals, Switzerland) over night at roomtemperature. After incubation with secondary antibody (HRP-Goat anti-ratantibody, Invitrogen, USA), enzyme-substrate reactions were performedusing 3, 3′-diaminobenzidine/H2O2 solution (Nichirei Bioscience Inc.,Japan).

For quantitative analysis of fibrosis, fat deposition and inflammationareas, bright field images of Sirius red-stained, oil red-stained andF4/80-immunostained sections were captured around the central vein usinga digital camera (DFC280; Leica, Germany) at 200-fold magnification, andthe positive areas in 5 fields/section were measured using ImageJsoftware (National Institute of Health, USA).

For quantitative PCR, Total RNA was extracted from liver samples usingRNAiso (Takara Bio Inc., Japan) according to the manufacturer'sinstructions. One μg of RNA was reverse-transcribed using a reactionmixture containing 4.4 mM MgCl2 (F. Hoffmann-La Roche Ltd.,Switzerland), 40 U RNase inhibitor (Toyobo Co., Ltd., Japan), 0.5 mMdNTP (Promega Corporation, USA), 6.28 μM random hexamer (PromegaCorporation), 5× first strand buffer (Promega), 10 mM dithiothreitol(Invitrogen) and 200 U MMLV-RT (Invitrogen) in a final volume of 20 μL.The reaction was carried out for 1 hour at 37° C., followed by 5 minutesat 99° C. Real-time PCR was performed using real-time PCR DICE and SYBRpremix Taq (Takara Bio). To calculate the relative mRNA expressionlevel, the expression of each gene was normalized to that of referencegene 36B4 (gene symbol: Rp1p0). PCR-primer sequences were as follows:36B4: forward 5′-TTCCAGGCTTTGGGCATCA-3′; reverse5′-ATGTTCAGCATGTTCAGCAGTGTG-3′; Alpha-SMA: forward5′-AAGAGCATCCGACACTGCTGAC-3′; reverse 5′-AGCACAGCCTGAATAGCCACATAC-3′;TIMP-1: forward 5′-TGAGCCCTGCTCAGCAAAGA-3′; reverse5′-GAGGACCTGATCCGTCCACAA-3′; Collagen Type 1: forward5′-CCAACAAGCATGTCTGGTTAGGAG-3′; reverse 5′-GCAATGCTGTTCTTGCAGTGGTA-3′;TGF-beta: MA030397 forward 5′-GTGTGGAGCAACATGTGGAACTCTA-3′; reverse5′-TTGGTTCAGCCACTGCCGTA-3′. Gene identities are as follows: 36B4:Ribosomal protein, large, P0, Alpha-SMA: Musculus actin, alpha 2, smoothmuscle, aorta (Acta2), Timp-1: Musculus tissue inhibitor ofmetalloproteinase 1 (Timp1), transcript variant 1, Collagen Type 1:Musculus collagen, type I, alpha 2 (Col1a2), TGF-beta: transforminggrowth factor beta.

For the treatment period 5-9 weeks, statistical analyses were performedusing the Bonferroni Multiple Comparison Test using GraphPad Prism 4(GraphPad Software Inc., USA). For the treatment period 7-9 weeks, tStatistical analyses were performed using Student's t-test usingGraphPad Prism 4. P values <0.05 were considered statisticallysignificant. A trend or tendency toward statistical significance wasidentified when a one-tailed t-test returned P values <0.10. Resultswere expressed as mean±standard deviation (SD).

Each study group contained 8 STAM mice treated subcutaneously witheither vehicle or PEG-Compound 1 in a volume of 1 mL/kg twice per week.Animals were sacrificed at 9 weeks. Study groups were as follows:

Group 1: Vehicle. Eight NASH mice were subcutaneously administeredvehicle in a volume of 1 mL/kg twice per week from 5 to 9 week of age.

Group 2: PEG-Compound 1-low dose. Eight NASH mice were subcutaneouslyadministered vehicle supplemented with PEG-Compound 1 at a dose of 1mg/kg twice per week from 5 to 9 weeks of age.

Group 3: PEG-Compound 1-high dose. Eight NASH mice were subcutaneouslyadministered vehicle supplemented with PEG-Compound 1 at a dose of 3mg/kg twice per week from 5 to 9 weeks of age.

Group 4: Vehicle. Eight NASH mice were subcutaneously administeredvehicle in a volume of 1 mL/kg twice per week from 7 to 9 week from ofage.

Group 5: PEG-Compound 1-high dose. Eight NASH mice were subcutaneouslyadministered vehicle supplemented with PEG-Compound 1 at a dose of 3mg/kg twice per week from 7 to 9 weeks of age.

The viability, clinical signs and behavior were monitored daily. Bodyweight was recorded before the treatment. Mice were observed forsignificant clinical signs of toxicity, moribundity and mortalityapproximately 60 minutes after each administration. At the end of thestudy, the animals were sacrificed by exsanguination through directcardiac puncture under ether anesthesia (Wako Pure Chemical Industries).

Results

Body weight changes are shown in FIG. 19.

For the treatment period 5-9 weeks (groups 1, 2, and 3) body weightgradually increased during the treatment period. Mean body weight of thePEG-Compound 1-low group was significantly lower than that of thevehicle group at day 11, 13, 15, 16, 18, 19, 20 and from day 22 to day28. Mean body weight of the PEG-Compound 1-high group was significantlylower than that of the vehicle group from day 10 to day 28. None of theanimals showed deterioration in general condition.

For the treatment period 7-9 weeks (groups 4 and 5) body weightgradually increased during the treatment period. Mean body weight of thePEG-Compound 1-high group was significantly lower than that of thevehicle group at day 5, 6, 9 and from day 11 to day 14. None of theanimals showed deterioration in general condition.

Total food consumption is shown in FIG. 20.

For the treatment period 5-9 weeks (groups 1, 2, and 3), there were nosignificant differences in total food consumption between the vehiclegroup and the PEG-Compound 1 treatment groups (Vehicle: 130±4 g/mouse,PEG-Compound 1-low: 130±3 g/mouse, PEG-Compound 1-high: 131±3 g/mouse).

For the treatment period 7-9 weeks (groups 4 and 5), the total foodconsumption tended to increase in the PEG-Compound 1-high group comparedwith the vehicle group (Vehicle: 62±3 g/mouse, PEG-Compound 1-high: 68±2g/mouse).

Body weight at the time of sacrifice is shown in FIG. 21 and Table 10.

For the treatment period 5-9 weeks (groups 1, 2, and 3), at the time ofsacrifice the PEG-Compound 1 treatment groups showed a significantdecrease in mean body weight compared with the vehicle group (Vehicle:21.7±1.4 g, PEG-Compound 1-low: 19.5±1.4 g, PEG-Compound 1-high:18.9±1.6 g).

For the treatment period 7-9 weeks (groups 4 and 5), at the time ofsacrifice the PEG-Compound 1-high group showed a significant decrease inmean body weight compared with the vehicle group (Vehicle: 21.5±1.4 g,PEG-Compound 1-high: 19.8±1.0 g).

Liver weight and liver-to-body weight ratio are shown in FIG. 22 andFIG. 23 and Table 10).

For the treatment period 5-9 weeks (groups 1, 2, and 3), thePEG-Compound 1 treatment groups showed a significant decrease in meanliver weight compared with the vehicle group (Vehicle: 1511±66 mg,PEG-Compound 1-low: 1163±117 mg, PEG-Compound 1-high: 970±237 mg). ThePEG-Compound 1 treatment groups showed a significant decrease in meanliver-to-body weight ratio compared with the vehicle group (Vehicle:7.0±0.4%, PEG-Compound 1-low: 6.0±0.5%, PEG-Compound 1-high: 5.1±0.9%).

For the treatment period 7-9 weeks (groups 4 and 5), the PEG-Compound1-high group showed a significant decrease in mean liver weight comparedwith the vehicle group (Vehicle: 1364±126 mg, PEG-Compound 1-high:1008±135 mg). The PEG-Compound 1-high group showed a significantdecrease in mean liver-to-body weight ratio compared with the vehiclegroup (Vehicle: 6.4±0.5%, PEG-Compound 1-high: 5.1±0.8%).

TABLE 10 Body weight and liver weight. PEG- PEG- PEG- Compound 1-Compound 1- Compound 1- Vehicle low high Vehicle high (5-9 wks (5-9 wks(5-9 wks (7-9 wks (7-9 wks Parameter treatment) treatment) treatment)treatment) treatment) (mean ± SD) (n = 8) (n = 8) (n = 8) (n = 8) (n =8) Body weight 21.7 ± 1.4 19.5 ± 1.4 18.9 ± 1.6  21.5 ± 1.4 19.8 ± 1.0(g) Liver weight 1511 ± 66  1163 ± 117 970 ± 237 1364 ± 126 1008 ± 135(mg) Liver-to-body  7.0 ± 0.4  6.0 ± 0.5 5.1 ± 0.9  6.4 ± 0.5  5.1 ± 0.8weight ratio (%)

Whole blood glucose is shown in FIG. 24 and Table 11.

For the treatment period 5-9 weeks (groups 1, 2, and 3), thePEG-Compound 1-high group showed a significant decrease in mean wholeblood glucose compared with the vehicle group. The blood glucose levelstended to decrease in the PEG-Compound 1-low group compared with thevehicle group (Vehicle: 691±114 mg/dL, PEG-Compound 1-low: 566±119mg/dL, PEG-Compound 1-high: 493±136 mg/dL).

For the treatment period 7-9 weeks (groups 4 and 5), the PEG-Compound1-high group showed a significant decrease in mean whole blood glucosecompared with the vehicle group (Vehicle: 656±85 mg/dL, PEG-Compound1-high: 454±104 mg/dL).

Plasma ALT is shown in FIG. 25 and Table 11.

For the treatment period 5-9 weeks (groups 1, 2, and 3), there were nosignificant differences in plasma ALT levels between the vehicle groupand the PEG-Compound 1 treatment groups (Vehicle: 82±66 U/L,PEG-Compound 1-low: 64±36 U/L, PEG-Compound 1-high: 108±78 U/L).

For the treatment period 7-9 weeks (groups 4 and 5), the plasma ALTlevels tended to decrease in the PEG-Compound 1-high group compared withthe vehicle group (Vehicle: 229±285 U/L, PEG-Compound 1-high: 36±8 U/L).

Plasma triglyceride levels are shown in FIG. 26 and Table 11.

For the treatment period 5-9 weeks (groups 1, 2, and 3), thePEG-Compound 1 treatment groups showed a significant decrease in plasmatriglyceride levels compared with the vehicle group (Vehicle: 322±341mg/dL, PEG-Compound 1-low: 75±39 mg/dL, PEG-Compound 1-high: 64±22mg/dL).

For the treatment period 7-9 weeks (groups 4 and 5), the PEG-Compound1-high group showed a significant decrease in plasma triglyceride levelscompared with the vehicle group (Vehicle: 139±39 mg/dL, PEG-Compound1-high: 59±51 mg/dL).

Plasma total cholesterol levels are shown in FIG. 27 and Table 11.

For the treatment period 5-9 weeks (groups 1, 2, and 3), the plasmatotal cholesterol levels tended to decrease in the PEG-Compound 1-highgroup compared with the vehicle group. There were no significantdifferences in plasma total cholesterol levels between the vehicle groupand the PEG-Compound 1-low group (Vehicle: 121±20 mg/dL, PEG-Compound1-low: 114±19 mg/dL, PEG-Compound 1-high: 98±31 mg/dL).

For the treatment period 7-9 weeks (groups 4 and 5), there were nosignificant differences in plasma total cholesterol levels between thevehicle group and the PEG-Compound 1-high group (Vehicle: 121±19 mg/dL,PEG-Compound 1-high: 115±29 mg/dL).

Liver triglyceride content is shown in FIG. 28 and Table 11.

For the treatment period 5-9 weeks (groups 1, 2, and 3), thePEG-Compound 1 treatment groups showed a significant decrease in livertriglyceride contents compared with the vehicle group (Vehicle: 54±13mg/g liver, PEG-Compound 1-low: 25±7 mg/g liver, PEG-Compound 1-high:22±9 mg/g liver).

For the treatment period 7-9 weeks (groups 4 and 5), the PEG-Compound1-high group showed a significant decrease in liver triglyceridecontents compared with the vehicle group (Vehicle: 53±11 mg/g liver,PEG-Compound 1-high: 17±6 mg/g liver).

Liver cholesterol content is shown in FIG. 29 and Table 11.

For the treatment period 5-9 weeks (groups 1, 2, and 3), there were nosignificant differences in liver cholesterol contents between thevehicle group and the PEG-Compound 1 treatment groups (Vehicle: 2.9±0.7mg/g liver, PEG-Compound 1-low: 2.9±0.6 mg/g liver, PEG-Compound 1-high:3.1±0.4 mg/g liver).

For the treatment period 7-9 weeks (groups 4 and 5), there were nosignificant differences in liver cholesterol contents between thevehicle group and the PEG-Compound 1 treatment groups (Vehicle: 3.1±0.8mg/g liver, PEG-Compound 1-high: 3.6±1.5 mg/g liver).

TABLE 11 Blood and liver biochemistry. PEG- PEG- PEG- Com- Com- Com-pound pound pound Vehicle 1-low 1-high Vehicle 1-high (5-9 wks (5-9 wks(5-9 wks (7-9 wks (7-9 wks Parameter treatment) treatment) treatment)treatment) treatment) (mean ± SD) (n = 8) (n = 8) (n = 8) (n = 8) (n =8) Whole blood 691 ± 114 566 ± 119 493 ± 136 656 ± 85  454 ± 104 glucose(mg/dL) Plasma ALT 82 ± 66 64 ± 36 108 ± 78  229 ± 285 36 ± 8  (U/L)Plasma 322 ± 341 75 ± 39 64 ± 22 139 ± 39  59 ± 51 triglyceride (mg/dL)Plasma total 121 ± 20  114 ± 19  98 ± 31 121 ± 19  115 ± 29  cholesterol(mg/dL) Liver 54 ± 13 25 ± 7  22 ± 9  53 ± 11 17 ± 6  triglyceride (mg/gliver) Liver 2.9 ± 0.7 2.9 ± 0.6 3.1 ± 0.4 3.1 ± 0.8 3.6 ± 1.5cholesterol (mg/g liver)

Histological Analyses: HE Staining and NAFLD Activity Score

Representative photomicrographs of the HE-stained sections are shown inFIG. 30A-B, and results of NAFLD Activity scores are shown in FIG. 31,FIG. 32A-C and Table 12. (The NALFD Activity Score (FIG. 31) is acomposite of steatosis, hepatocyte ballooning, and lobular inflammationscores). The results shown in Table 12 were based upon the scoringcriteria shown in Table 13.

TABLE 12 NAFLD Activity score. Score Lobular Hepatocyte NAS SteatosisInflammation Ballooning (mean ± Group n 0 1 2 3 0 1 2 3 0 1 2 SD)Vehicle 8 — 6 2 — — — 5 3 — — 8 5.6 ± 0.7 (5-9 wks treatment) PEG- 8 3 5— — — 4 4 — — 1 7 4.0 ± 1.2 Compound 1-low (5-9 wks treatment) PEG- 8 44 — — — 5 3 — 2 4 2 2.9 ± 1.2 Compound 1-high (5-9 wks treatment)Vehicle 8 — 5 3 — — — 6 2 — — 8 5.6 ± 0.7 (7-9 wks treatment) PEG- 8 5 3— — — 6 2 — 3 4 1 2.4 ± 1.3 Compound 1-high (7-9 wks treatment)

TABLE 13 Definition of NAS Components Item Score Extent Steatosis 0    <5% 1  5-33% 2 >33-66% 3    >66% Hepatocyte Ballooning 0 None 1 Fewballoon cells 2 Many cells/prominent ballooning Lobular Inflammation 0No foci 1 <2 foci/200x 2 2-4 foci/200x  3 >4 foci/200x

For the treatment period 5-9 weeks (groups 1, 2, and 3), liver sectionsfrom the vehicle group exhibited severe micro- and macrovesicular fatdeposition, hepatocellular ballooning and inflammatory cellinfiltration. The PEG-Compound 1 treatment groups showed markedimprovements (decreases) in fat deposition, hepatocellular ballooningand inflammatory cell infiltration, with significant reduction in NAFLDActivity score (NAS) compared with the vehicle group (Vehicle: 5.6±0.7,PEG-Compound 1-low: 4.0±1.2, PEG-Compound 1-high: 2.9±1.2).

For the treatment period 7-9 weeks (groups 4 and 5), the PEG-Compound1-high group showed marked improvements (decreases) in fat deposition,hepatocellular ballooning and inflammatory cell infiltration, withsignificant reduction in NAS compared with the vehicle group (Vehicle:5.6±0.7, PEG-Compound 1-high: 2.4±1.3).

Histological Analyses: Sirius Red Staining

Results of Sirius red staining are shown in FIG. 33A-B, FIG. 34 (showingrepresentative staining) and Table 14.

For the treatment period 5-9 weeks (groups 1, 2, and 3), liver sectionsfrom the vehicle group exhibited collagen deposition in the pericentralregion of the liver lobule. The fibrosis area (Sirius red-positive area)significantly decreased in the PEG-Compound 1 treatment groups comparedwith the vehicle group (Vehicle: 1.10±0.24%, PEG-Compound 1-low:0.71±0.17%, PEG-Compound 1-high: 0.71±0.19%).

For the treatment period 7-9 weeks (groups 4 and 5), the fibrosis areasignificantly decreased in the PEG-Compound 1-high group compared withvehicle group (Vehicle: 1.25±0.29%, PEG-Compound 1-high: 0.75±0.21%).

Histological Analyses: F4/80 Immunohistochemistry

Result of F4/80 immunohistochemistry are shown in FIG. 35A-B, FIG. 36(showing representative staining) and Table 14.

For the treatment period 5-9 weeks (groups 1, 2, and 3), F4/80immunostaining of liver sections form the vehicle group demonstratedaccumulation of F4/80+ cells in the liver lobule. There were nosignificant differences in the number and size of F4/80+ cells betweenthe vehicle group and the PEG-Compound 1 treatment groups, as well as inthe percentage of inflammation area (F4/80-positive area) (Vehicle:6.9±0.9%, PEG-Compound 1-low: 7.6±1.5%, PEG-Compound 1-high: 7.1±0.7%).

For the treatment period 7-9 weeks (groups 4 and 5), there were nosignificant differences in the number and size of F4/80+ cells betweenthe vehicle group and the PEG-Compound 1-high group, as well as in thepercentage of inflammation area (Vehicle: 7.1±0.7%, PEG-Compound 1-high:6.3±1.1%).

Histological Analysis: Oil Red Staining

Results of oil red staining are shown in FIG. 37A-B (showingrepresentative staining), FIG. 38 and Table 14.

For the treatment period 5-9 weeks (groups 1, 2, and 3), liver sectionsfrom the vehicle group exhibited micro- and macrovesicular fatdeposition in the hepatocytes. The percentage of fat deposition area(oil red-positive area) significantly decreased in the PEG-Compound 1treatment groups compared with the vehicle group (Vehicle: 30.4±4.8%,PEG-Compound 1-low: 20.5±8.8%, PEG-Compound 1-high: 16.2±6.1%).

For the treatment period 7-9 weeks (groups 4 and 5), the percentage offat deposition area significantly decreased in the PEG-Compound 1-highgroup compared with the vehicle group (Vehicle: 30.7±5.6%, PEG-Compound1-high: 13.9±7.7%).

TABLE 14 Histological analyses PEG-Com- PEG-Com- PEG-Com- Vehicle pound1-low pound 1-high Vehicle pound 1-high (5-9 wks (5-9 wks (5-9 wks (7-9wks (7-9 wks Parameter treatment) treatment) treatment) treatment)treatment) (mean ± SD) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) Siriusred- 1.10 ± 0.24 0.71 ± 0.17 0.71 ± 0.19 1.25 ± 0.29 0.75 ± 0.21positive area (%) F4/80-positive 6.9 ± 0.9 7.6 ± 1.5 7.1 ± 0.7 7.1 ± 0.76.3 ± 1.1 area (%) Oil red- 30.4 ± 4.8  20.5 ± 8.8  16.2 ± 6.1  30.7 ±5.6  13.9 ± 7.7  positive area (%)

Gene Expression Analyses

Results of gene expression analysis are shown in FIG. 39A-D and Table 15for alpha-SMA, TIMP-1, Collagen Type 1, and TGF-beta.

For the treatment period 5-9 weeks (groups 1, 2, and 3), alpha-SMA mRNAexpression levels tended to be down-regulated in the PEG-Compound 1-highgroup compared with the vehicle group. There were no significantdifferences in α-SMA mRNA expression levels between the vehicle groupand the PEG-Compound 1-low group (Vehicle: 1.00±1.14, PEG-Compound1-low: 0.52±0.60, PEG-Compound 1-high: 0.39±0.30).

For the treatment period 7-9 weeks (groups 4 and 5), there were nosignificant differences in α-SMA mRNA expression levels between thevehicle group and the PEG-Compound 1-high group (Vehicle: 1.00±0.88,PEG-Compound 1-high: 1.14±1.01).

For the treatment period 5-9 weeks (groups 1, 2, and 3), TIMP-1 mRNAexpression levels tended to be down-regulated in the PEG-Compound 1-lowgroup compared with the vehicle group. There were no significantdifferences in TIMP-1 mRNA expression levels between the vehicle groupand the PEG-Compound 1-high group (Vehicle: 1.00±0.39, PEG-Compound1-low: 0.73±0.28, PEG-Compound 1-high: 0.88±0.31).

For the treatment period 7-9 weeks (groups 4 and 5), TIMP-1 mRNAexpression levels tended to be down-regulated in the PEG-Compound 1-highgroup compared with the vehicle group (Vehicle: 1.00±0.57, PEG-Compound1-high: 0.65±0.34).

For the treatment period 5-9 weeks (groups 1, 2, and 3), there were nosignificant differences in Collagen Type 1 mRNA expression levelsbetween the vehicle group and the PEG-Compound 1 treatment groups(Vehicle: 1.00±0.22, PEG-Compound 1-low: 0.88±0.24, PEG-Compound 1-high:0.89±0.34).

For the treatment period 7-9 weeks (groups 4 and 5), the Collagen Type 1mRNA expression significantly decreased in the PEG-Compound 1-high groupcompared with the vehicle group (Vehicle: 1.00±0.29, PEG-Compound1-high: 0.69±0.20).

For the treatment period 5-9 weeks (groups 1, 2, and 3), TGF-β mRNAexpression levels tended to be down-regulated in the PEG-Compound 1-lowgroup compared with the vehicle group. There were no significantdifferences in TGF-β mRNA expression levels between the vehicle groupand the PEG-Compound 1-high group (Vehicle: 1.00±0.24, PEG-Compound1-low: 1.07±0.60, PEG-Compound 1-high: 0.90±0.26).

For the treatment period 7-9 weeks (groups 4 and 5), TGF-β mRNAexpression levels tended to be down-regulated in the PEG-Compound 1-highgroup compared with the vehicle group (Vehicle: 1.00±0.45, PEG-Compound1-high: 0.77±0.22).

TABLE 15 Gene expression analyses PEG-Com PEG-Com PEG-Com Vehicle pound1-low pound 1-high Vehicle pound 1-high (5-9 wks (5-9 wks (5-9 wks (7-9wks (7-9 wks Parameter treatment) treatment) treatment) treatment)treatment) (mean ± SD) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) Alpha-SMA1.00 ± 1.14 0.52 ± 0.60 0.39 ± 0.30 1.00 ± 0.88 1.14 ± 1.01 TIMP-1 1.00± 0.39 0.73 ± 0.28 0.88 ± 0.31 1.00 ± 0.57 0.65 ± 0.34 Collagen Type1.00 ± 0.22 0.88 ± 0.24 0.89 ± 0.34 1.00 ± 0.29 0.69 ± 0.20 1 TGF-β 1.00± 0.24 1.07 ± 0.60 0.90 ± 0.26 1.00 ± 0.45 0.77 ± 0.22

As noted above, PEG-Compound 1 reduced hepatic fat accumulation asassessed by a biochemical assay measuring hepatic triglyceride contentand histology following staining of liver sections with hematoxylin andeosin or oil red O. This anti-steatotic activity of native FGF21 hasbeen reported in the literature to depend on adiponectin in the mouse(see Lin et al., Cell Metab. 17: 779-789 (2013); Holland et al., CellMetab 17: 790-797 (2013), each of which is hereby incorporated byreference in its entirety). Therefore, concentrations of totaladiponectin were measured in terminal serum samples prepared from thetreated mice. Serum adiponectin was measured following themanufacturer's protocol using a commercially available ELISA kit (Alpcocatalog number 47-ADPMS-E01).

Twice weekly administration of 3 mg/kg PEG-Compound 1 statisticallysignificantly increased serum total adiponectin, as compared to thecorresponding vehicle group, at all terminal time-points tested (FIG.61). This result is consistent with the hypothesis that adiponectincontributes to the efficacy of PEG-Compound 1 in the Stelic NASH model.

Discussion

This example shows the results of experiments that tested the efficacyof PEG-Compound 1 in both a preventative and a therapeutic model ofNASH. In the preventative model, treatment was initiated at the timefatty liver was evident in this model (starting from week 5, i.e., week5-9 groups). In the therapeutic model, treatment was initiated at thetime NASH was evident in this model (starting from week 7, i.e., week7-9 groups). Thus, therapeutic efficacy of PEG-Compound 1 wasdemonstrated in both the preventative and therapeutic models.

Treatment with PEG-Compound 1 significantly reduced the fibrosis area(detected by Sirius red staining) in both treatment models,demonstrating the anti-fibrotic effect of PEG-Compound 1. PEG-Compound 1treatment also reduced the mRNA expression levels of α-SMA, TIMP-1 andCollagen Type 1, further supporting the anti-fibrotic properties ofPEG-Compound 1. Treatment with PEG-Compound 1 also caused statisticallysignificant decreases in body and liver weight, liver-to-body weightratio, blood glucose, plasma and liver triglycerides, NAFLD activityscore, fat deposition area (detected by oil red staining).

All PEG-Compound 1 treatment groups showed a significant reduction ofNAS, which is one of the clinical endpoints for assessing the activityof NASH (Sanyal A J. et al., Hepatology, 2011; 54:344).

Furthermore, treatment with PEG-Compound 1 improved lipid and glucosemetabolism as evidenced by reduction of whole blood glucose levels,plasma triglyceride levels and liver triglyceride contents.

In conclusion, PEG-Compound 1 showed anti-NASH, anti-fibrotic effectsassociated with improved lipid and glucose metabolism in the presentstudy.

Example 14 Solubility Assessment of Modified FGF-21 PolypeptidesComprising a Fusion Partner

This example describes measurement of the relative solubility ofmodified FGF-21 polypeptides comprising a fusion partner. The resultsare indicative of the ability of a compound to be formulated to arelatively higher concentration, which would permit more facileadministration of an effective dosage.

Methods

Relative solubility assessments were performed by sequential plug-flowconcentration cycles followed by size-exclusion chromatography analysis.Samples, formulated in 20 mM Histidine buffer pH 7.0 at similar but notidentical starting concentrations, were pipetted into 3 kDa molecularweight cut-off centrifuge concentrators and spun at 4,750 RPM for 15minute, 15 minute, and 40 minute cycles at 4° C. In between spin cycles,aliquots were removed from the concentration apparatus and analyticalsize exclusion chromatography analysis (aSEC) was performed (on a GEHealthcare Superdex S-75 10/300 GL column equilibrated in PBS pH 7.2buffer) to determine the concentration of HMW and monomer species in thesolution.

Total Concentrations were determined by absorbance at 280 nm with aNanoDrop spectrophotometer. High molecular weight percentage wasdetermined by area under the curve calculations of high molecular weightpeaks relative to the monomer peaks in the SEC chromatogram trace. Theresulting data points are plotted to visualize the rank order of leastsoluble constructs to most soluble under the conditions tested, thelower the slope created by the data points the more stable the proteinvariant under the conditions tested

Results

Relative solubility of Modified FGF-21 polypeptides comprising a fusionpartner was determined by measuring the formation of high molecularweight (HMW) aggregates as a function of protein concentration. LowerHMW aggregate formation at a given concentration is indicative ofgreater solubility, which would result in the ability to be formulatedto a higher concentration.

Results are shown graphically in FIG. 41. The slope of the plug flowsolubility curve for the tested compounds is shown below in Table 16(lower values indicate less aggregate formation and hence the ability tobe formulated to a higher concentration).

TABLE 16 Plug Flow Solubility Results for modified FGF-21 Polypeptidescomprising a fusion partner FGF21- PKE Adnectin(2) Plug Flow SolubilityFusion Compound Slope Compound 101 2.72 Compound 102 0.31 Compound 103n.d. Compound 104 0.36 Compound 105 0.88 Compound 106 0.83 Compound 1070.37 Compound 108 0.68 Compound 109 0.76 Compound 110 0.25 Compound 1110.65 Compound 112 0.05 Compound 113 0.47

Example 15 A Randomized, Double-Blind, Placebo-Controlled, ParallelGroup, Multiple Dose Study to Evaluate the Safety, Pharmacokinetics andPharmacodynamic Effects of PEG-Compound 1 in Adults with Non-AlcoholicSteatohepatitis

In the United States, NASH is one of the leading causes of cirrhosis inadults; up to 20% of adults with NASH develop cirrhosis. Thehistological findings of NASH include steatosis, inflammation andballooning degeneration with varying amounts of pericellular fibrosis inliver, and occur in the absence of significant alcohol use. NASHpatients exhibit increased mortality rates from cardiovascular-, liver-and cancer-related deaths. No specific medicinal treatment options areavailable.

This example describes a randomized, double-blind and placebo-controlledhuman clinical trial to evaluate the safety, pharmacokinetics andpharmacodynamic effects of PEG-Compound 1 (polypeptide of SEQ ID NO:201, where pAF is linked to a 30 kDa PEG) in adults with NASH, includingevaluation of safety, tolerability and change in hepatic fat fraction(%) by MRI.

Study Population:

Approximately 90 male and female subjects aged 21 to 75 years areenrolled meeting all of the following criteria: a liver biopsy performedwithin 1 year of screening with documented results of NASH with NASH CRNfibrosis stage 1-3 or equivalent using a different scoring system; a BMIof ≧30 (weight (kg)/[height (m)]²); a fatty liver index ≧60; a hepaticfat fraction (%) ≧10% by MRI performed during the screening period.Subjects undergo screening evaluations to determine eligibility within42 days prior to randomization.

Subjects are excluded from the study if there is evidence of concurrentdisease including chronic liver disease (other than NASH), cirrhosis,decompensated liver disease, uncontrolled diabetes, and certain othermedical conditions or history.

Baseline scans are conducted approximately 14 to 35 days prior to theDay 1 dose of the active drug or placebo, including hepatic fat contentby magnetic resonance imaging (MRI), liver stiffness by magneticresonance elastography (MRE), and body composition by dual-energy X-rayabsorptiometry (DXA). Additional baseline patient evaluation includesdetermining weight, BMI, and waist circumference.

Treatment Regimen:

PEG-Compound 1 is administered daily or weekly for 16 weeks to adultswith NASH. Treatment is self-administered subcutaneously subsequent to7-day patient training in proper use of the injector with placebo.Approximately 90 eligible subjects are randomized on Day 1 to one ofthree treatment groups (30 per group), who are then treated (startingfrom Day 1) as follows: Treatment A: 10 mg PEG-Compound 1 daily;Treatment B: 20 mg PEG-Compound 1 weekly; Treatment C: Placebo daily.Subjects are stratified using diagnosis of Type 2 diabetes mellitus (yesvs no) based on current American Diabetes Association criteria.

All subjects self-administer PEG-Compound 1 or placebo once daily.PEG-Compound 1 is provided in 10 mg/mL solution. For the Treatment B (20mg/wk) group, 2 injections of 1 mL each are given concurrently on Day 1(D 1) and on Days 2-7, the injection is placebo. To maintain blind theother two groups also have two Day 1 injections, one or both of whichcontain a placebo. On Days 2-7, the injection is 1 mL of PEG-Compound 1(Treatment A group) or 1 mL placebo (Treatment C group). Treatmentcontinues until day 112 (D 112) (i.e., 16 weeks).

Patient Evaluation:

Patient evaluation is conducted at D −7 (the start of the placebo-onlylead-in), D 1, D 15, D 29, D 43, D 57, D 86, and D 112. Physicalexaminations, vital sign measurements, 12-lead electrocardiograms (ECG),clinical laboratory evaluations, and MRI, MRE and DXA scans areperformed. The MRI, MRE and DXA end-of-treatment scans are conducted atDay 112 (+/−1 week). Follow-up visits are conducted at around D 142 andD 292, with the DXA follow-up scanning conducted 6 months (+/−2 weeks)after the last dose. Blood is collected for pharmacokinetic (PK) andpharmacodynamic (PD) analysis. Serum concentration of PEG-Compound 1(Total and C-terminal intact) are measured by a validated assay. All PKdata collected in the study are assessed by a developed population PKmodel to estimate model-based parameters such as CL/F, Vc/F, Ka etc.Furthermore, estimates of individual exposure parameters (such as Cavg,Cmin, Cmax and AUC at steady state) are derived from the model-basedparameters. Subjects are closely monitored for adverse events throughoutthe study.

Primary Endpoints:

The primary objective is to assess the effect of daily or weekly dosesof PEG-Compound 1 on safety, tolerability and hepatic fat fraction (%)by MRI in patients with biopsy proven NASH. This will be assessed by theprimary endpoint of change in percent hepatic fat fraction (%) frombaseline to Week 16, determined by proton density fat-fraction hepaticMRI. Baseline is defined as the last non-missing pre-dose measurementfor all the endpoints. PEG-Compound 1 administered daily or weekly for16 weeks to patients with NASH is predicted to lower hepatic fatfraction (%) to a greater extent than placebo. Primary endpoints alsoinclude safety endpoints including incidence of AEs, serious AEs, andevents of special interest including injection site assessment, AEsleading to discontinuation, and death as well as marked abnormalities inclinical laboratory tests, vital sign measurements, ECGs, physicalexaminations and bone mineral density (BMD) collected by DXA scan atspecified time points.

Secondary Endpoints:

Secondary endpoints include pharmacokinetic endpoints and immunogenicityendpoints. The pharmacokinetic endpoints will be assessed by model-basedpharmacokinetic parameters of PEG-Compound 1 (Total and C-Terminalintact) serum concentration: Cavg, Cmin, Cmax and AUC(TAU) determinedfrom measurements conducted at selected time points. Immunogenicityendpoints are assessed by patient levels of anti-PEG-Compound 1antibodies and anti-FGF21 antibodies. Model-based pharmacokineticparameters of PEG-Compound 1 (Total and C-Terminal intact) serumconcentration are determined. Primary PK parameters include: CL/F(apparent clearance after extra-venous administration); V/F (Apparentvolume of distribution after extra-venous administration); and Ka (rateconstant of absorption from injection site into blood circulation). Fromthe primary PK parameters, secondary PK parameters are derived,including Cmax (maximum calculated serum concentration); T½ (eliminationhalf-life); AUC(TAU) (area under the concentration-time curve in onedosing interval); Cmin (minimal concentration within dosing interval);and Cavg (average concentration within dosing interval).

Analysis:

A longitudinal repeated measures analysis is used to analyze the changein hepatic fat fraction (%) at Week 16 from baseline in the treatedpopulation who have both a baseline and at least one post-baselinemeasurement. The model includes treatment group, week andtreatment-by-week interactions as main effects and baseline hepatic fatfraction (%) and baseline diabetic status as covariates. An unstructuredcovariance matrix is used to represent the correlation of the repeatedmeasures within each subject. The model provides point estimates,standard errors and 2-sided 90% confidence intervals for mean changefrom baseline within and between treatments. P-values are calculated tocompare the treatment effect in each of two PEG-Compound 1 treatmentgroups (10 mg daily and 20 mg weekly) to that in the placebo treatmentgroup at Week 16. Each treatment group comparison is performed at aone-sided 0.05 significance level. No adjustments are made formultiplicity.

The relationship between PEG-Compound 1 (total and C-terminal intact)exposure and other biomarkers or endpoints are explored to showdose-response relationships. These measurements are also analyzed toshow their relationship to the change in hepatic fat fraction and howthese biomarkers predict or relate to that change. These endpoints andbiomarkers include changes in liver stiffness by MRE, body weight, BMI,waist circumference, body composition by DXA, Glucose homeostasis,insulin sensitivity, fasting lipids, bone homeostasis, ALT and AST, aswell as biomarkers associated with the risk of disease progression andcomplications. NASH is typically associated with reduced levels ofadiponectin (hypoadiponectinemia), and reduced levels are associatedwith more extensive necroinflammation. Adiponectin is believed toincrease insulin sensitivity by enhancing fat oxidation and reducinghepatic lipid storage. Serum total adiponectin levels are expected to beincreased by PEG-Compound 1 treatment. PEG-Compound 1 treatment is alsoexpected to reduce fasting triglyceride, LDL, ApoB, ApoC3 and increaseHDL in NASH patients.

Analysis of the study results, using an unstructured covariance matrixas described above, is conducted to show a statistically significant(p<0.05) reduction in hepatic fat fraction in patients treated withPEG-Compound 1 (in both the daily and weekly administration studygroups) compared to placebo controls.

Example 16

This example further explores the efficacy of PEG-Compound 1 in the STAMmodel of Non-alcoholic Steatohepatitis (NASH). Mice were treated between9 and 12 weeks of age or to 15 weeks of age.

Methods

PEG-Compound 1 and Vehicle (20 mM Tris/250 mM sucrose, pH 8.3) wereprovided. To prepare dosing solution, PEG-Compound 1 solution wasprepared by appropriate dilution with the provided vehicle.

C57BL/6 mice (15-day-pregnant female) were obtained from Japan SLC, Inc.(Shizuoka, Japan). All animals used in the study were housed and caredfor in accordance with the Japanese Pharmacological Society Guidelinesfor Animal Use. NASH was induced in 60 male mice by a singlesubcutaneous injection of 200 μg streptozotocin (STZ, Sigma-Aldrich,USA) solution 2 days after birth and feeding with high fat diet (HFD, 57kcal % fat, cat#: HFD32, CLEA Japan, Inc., Japan) after 4 weeks of age.

PEG-Compound 1 and Vehicle were administered by subcutaneously route ina volume of 1 mL/kg at a dose of 3 mg/kg twice per week. The animalswere maintained in a SPF facility under controlled conditions oftemperature (23±2° C.), humidity (45±10%), lighting (12-hour artificiallight and dark cycles; light from 8:00 to 20:00) and air exchange. Ahigh pressure (20±4 Pa) was maintained in the experimental room toprevent contamination of the facility. The animals were housed inpolycarbonate cages KN-600 (Natsume Seisakusho, Japan) with a maximum of4 mice per cage. Sterilized Paper-Clean (Japan SLC) was used for beddingand replaced once a week. Sterilized solid HFD was provided ad libitum,being placed in the metal lid on top of the cage. Pure water wasprovided ad libitum from a water bottle equipped with a rubber stopperand a sipper tube. Water bottles were replaced once a week, cleaned andsterilized in autoclave and reused. Mice were identified by numbersengraved on earrings. Each cage was labeled with a specificidentification code.

Non-fasting blood glucose was measured in whole blood using LIFE CHECK(EIDIA Co. Ltd., Japan). For plasma biochemistry, blood was collected inpolypropylene tubes with anticoagulant (Novo-Heparin, MochidaPharmaceutical Co. Ltd., Japan) and centrifuged at 1,000×g for 15minutes at 4° C. The supernatant was collected and stored at −80° C.until use. Plasma ALT, triglyceride and total cholesterol levels weremeasured by FUJI DRI-CHEM 7000 (Fujifilm Corporation, Japan).

Liver total lipid-extracts were obtained by Folch's method (Folch J. etal., J. Biol. Chem. 1957; 226: 497). Liver samples were homogenized inchloroform-methanol (2:1, v/v) and incubated overnight at roomtemperature. After washing with chloroform-methanol-water (8:4:3,v/v/v), the extracts were evaporated to dryness, and dissolved inisopropanol. Liver triglyceride and cholesterol contents were measuredby Triglyceride E-test and Cholesterol E-test, respectively (Wako PureChemical Industries, Ltd., Japan).

For HE staining, sections were cut from paraffin blocks of liver tissueprefixed in Bouin's solution and stained with Lillie-Mayer's Hematoxylin(Muto Pure Chemicals Co., Ltd., Japan) and eosin solution (Wako PureChemical Industries). NAFLD Activity score (NAS) was calculatedaccording to the criteria of Kleiner (Kleiner D E. et al., Hepatology,2005; 41:1313). To visualize collagen deposition, Bouin's fixed liversections were stained using picro-Sirius red solution (Waldeck,Germany). To visualize macro- and microvesicular fat, cryosections werecut from frozen liver tissues, prefixed in 10% neutral bufferedformalin, embedded in Tissue-Tek O.C.T. compound (Sakura Finetek JapanCo. Ltd., Japan), and stained with Oil Red O (Sigma-Aldrich). Forimmunohistochemistry, sections were cut from frozen liver tissuesembedded in Tissue-Tek O.C.T. compound and fixed in acetone. Endogenousperoxidase activity was blocked using 0.03% H2O2 for 5 minutes, followedby incubation with Block Ace (Dainippon Sumitomo Pharma Co. Ltd., Japan)for 10 minutes. The sections were incubated with a 200-fold dilution ofanti-F4/80 antibody (BMA Biomedicals, Switzerland) over night at 4° C.After incubation with secondary antibody (HRP-Goat anti-rat antibody,Invitrogen, USA), enzyme-substrate reactions were performed using 3,3′-diaminobenzidine/H2O2 solution (Nichirei Bioscience Inc., Japan).

The kidney was fixed in Bouin's solution. The glomerular architecturewas observed using PAS staining with sections oxidized by 0.5% periodicacid and stained with Schiff s reagent (both from Wako Pure ChemicalIndustries). To visualize collagen deposition, kidney sections werestained with picro-Sirius red solution (Waldeck).

For quantitative analysis of fibrosis, fat deposition and inflammationareas bright field images of Sirius red-stained, oil red-stained andF4/80-immunostained sections were captured around the central vein forlivers or interstitial region for kidneys using a digital camera(DFC280; Leica, Germany) at 200-fold magnification, and the positiveareas in 5 fields/section were measured using ImageJ software (NationalInstitute of Health, USA).

Statistical analyses were performed using Bonferroni Multiple ComparisonTest on GraphPad Prism 4 (GraphPad Software Inc., USA). P values <0.05were considered statistically significant. A trend or tendency wasassumed when a one-tailed t-test returned P values <0.05. Results wereexpressed as mean±SD.

Results

Study Groups were as follows (summarized in Table 17 below).

Group 1: Vehicle. Twenty NASH mice were subcutaneously administeredvehicle in a volume of 1 mL/kg twice per week from 9 to 12 weeks of ageor to 15 weeks of age.

Group 2: PEG-Compound 1. Twenty NASH mice were subcutaneouslyadministered vehicle supplemented with PEG-Compound 1 at a dose of 3mg/kg twice per week from 9 to 12 weeks of age or to 15 weeks of age.

TABLE 17 Treatment summary. Test Dose Volume Sacrifice Group No. miceMice substance (mg/kg) (mL/kg) Regimens (wks) 1 20 STAM Vehicle — 1 SC,twice per 12, 15 weeks, 9 wks-12 wks or 15 wks 2 20 STAM PEG- 3 1 SC,twice per 12, 15 Compound 1 weeks, 9 wks-12 wks or 15 wks

The viability, clinical signs and behavior were monitored daily. Bodyweight was recorded before the treatment. Mice were observed forsignificant clinical signs of toxicity, moribundity and mortalityapproximately 60 minutes after each administration. The animals weresacrificed by exsanguination through direct cardiac puncture under etheranesthesia (Wako Pure Chemical Industries). Six animals in all groupswere sacrificed at 12 weeks of age for the following assays, and theremaining animals were kept until the day of sacrifice at 15 weeks ofage.

Body weight in the all groups did not obviously change during thetreatment period. There were no significant differences in mean bodyweight between the Vehicle group and the PEG-Compound 1 group.

During the treatment period, mice died before reaching day 40 asfollows: eight out of 20 mice died in the Vehicle group. No animals werefound dead in the PEG-Compound 1 group. The cause of the death remainedunclear and could be disease progression related in the Vehicle group.The days of death of the individual mice were days 7, 17 (two mice), 20(two mice), 34, 38, and 40. After 5 mice died in the Vehicle group, thedecision was made to modify the original study protocol and reduce thenumber of mice sacrificed at week 12 from 8 to 6 per group in an attemptto ensure that at least 6 mice in each group would survive to week 15.

In the animals treated between weeks 9-12, there were no significantdifferences in the mean body weight on the day of sacrifice between theVehicle group and the PEG-Compound 1 group (FIG. 42A). Likewise, in theanimals treated between weeks 9-15, there were no significantdifferences in the mean body weight on the day of sacrifice between theVehicle group and the PEG-Compound 1 group (FIG. 42B).

Organ weight and liver-to-body weight ratio are shown in FIGS. 43-46 andsummarized in Table 18.

In the animals treated between weeks 9-12, the PEG-Compound 1 groupshowed decreasing tendencies in mean liver weight compared with theVehicle group (FIG. 43A). The mean liver-to-body weight ratiosignificantly decreased in the PEG-Compound 1 group compared with theVehicle group (p<0.01) (FIG. 44A). There were no significant differencesin the mean right kidney weight between the Vehicle group and thePEG-Compound 1 group (FIG. 45A). Mean left kidney weight tended todecrease in the PEG-Compound 1 group compared with the Vehicle group(FIG. 46A).

In the animals treated between weeks 9-15, the PEG-Compound 1 groupsignificantly decreased mean liver weight (p<0.001) (FIG. 43B) and meanliver-to-body weight ratio (p<0.001) (FIG. 44B) compared with theVehicle group. There were no significant differences in the mean rightkidney weight (FIG. 45B) and mean left kidney weight (FIG. 46B) betweenthe Vehicle group and the PEG-Compound 1 group.

TABLE 18 Body weight and organ weight results. Parameter 12 wks Vehicle12 wks PEG-Compound 1 (mean ± SD) (n = 6) (n = 6) Body weight (g) 18.0 ±4.7 16.0 ± 3.9  Liver weight (mg) 1389 ± 374 973 ± 226 Liver-to-bodyweight  7.7 ± 1.1 6.1 ± 0.7 ratio (%) Right kidney weight (mg) 143 ± 40110 ± 25  Left kidney weight (mg) 148 ± 37 98 ± 26 Parameter 15 wksVehicle 15 wks PEG-Compound 1 (mean ± SD) (n = 6) (n = 14) Body weight(g) 16.5 ± 4.8 15.2 ± 2.5  Liver weight (mg) 1341 ± 374 746 ± 161Liver-to-body weight  8.2 ± 1.5 5.0 ± 1.1 ratio (%) Right kidney weight(mg) 111 ± 26 97 ± 24 Left kidney weight (mg) 101 ± 22 90 ± 20

Biochemical measurement results are shown in FIGS. 47-52 and summarizedin Table 19, below.

In the animals treated between weeks 9-12, there were no significantdifferences in whole blood glucose levels between the Vehicle group andthe PEG-Compound 1 group (FIG. 47A).

In the animals treated between weeks 9-15, whole blood glucose levels inthe PEG-Compound 1 group significantly decreased compared with theVehicle group (p<0.05) (FIG. 47B).

In the animals treated between weeks 9-12, there was no significantdifference in plasma ALT levels between the Vehicle group and thePEG-Compound 1 group (FIG. 48A).

In the animals treated between weeks 9-15, plasma ALT levels in thePEG-Compound 1 group significantly decreased compared with the Vehiclegroup (p<0.01) (FIG. 48B).

In the animals treated between weeks 9-12, the PEG-Compound 1 groupshowed a tendency to decrease in plasma triglyceride levels comparedwith the Vehicle group (FIG. 49A).

In the animals treated between weeks 9-15, there was no significantdifference in plasma triglyceride levels between the Vehicle group andthe PEG-Compound 1 group (FIG. 49B).

In the animals treated between weeks 9-12, there were no significantdifferences in plasma total cholesterol between the Vehicle group andthe PEG-Compound 1 group (FIG. 50A).

In the animals treated between weeks 9-15, there were no significantdifferences in plasma total cholesterol between the Vehicle group andthe PEG-Compound 1 group (FIG. 50B).

In the animals treated between weeks 9-12, liver triglyceride contentsin the PEG-Compound 1 group significantly decreased compared with theVehicle group (p<0.01) (FIG. 51A).

In the animals treated between weeks 9-15, liver triglyceride contentsin the PEG-Compound 1 group significantly decreased compared with theVehicle group (p<0.001) (FIG. 51B).

In the animals treated between weeks 9-12, liver cholesterol contents inthe PEG-Compound 1 group significantly decreased compared with theVehicle group (p<0.01) (FIG. 52A).

In the animals treated between weeks 9-15, liver cholesterol contents inthe PEG-Compound 1 group significantly decreased compared with theVehicle group (p<0.001) (FIG. 52B).

TABLE 19 Biochemical test results. Parameter 12 wks Vehicle 12 wksPEG-Compound 1 (mean ± SD) (n = 6) (n = 6) Whole blood glucose 628 ± 268505 ± 231 (mg/dL) Plasma ALT (U/L) 55 ± 30 39 ± 21 Plasma triglyceride498 ± 368 167 ± 214 (mg/dL) Plasma total cholesterol 195 ± 115 162 ± 62 (mg/dL) Liver triglyceride 98 ± 38 42 ± 13 (mg/g liver) Livercholesterol 36 ± 15 18 ± 4  (mg/g liver) Parameter 15 wks Vehicle 15 wksPEG-Compound 1 (mean ± SD) (n = 6) (n = 14) Whole blood glucose 594 ±182 350 ± 174 (mg/dL) Plasma ALT (U/L) 48 ± 15 23 ± 15 Plasmatriglyceride 117 ± 24  102 ± 57  (mg/dL) Plasma total cholesterol 176 ±72  141 ± 45  (mg/dL) Liver triglyceride 98 ± 31 49 ± 19 (mg/g liver)Liver cholesterol 39 ± 10 19 ± 9  (mg/g liver)

Representative micrographs showing histological analysis of liversamples are shown in FIGS. 53, 55, 57, and 59. Summaries of histologicalresults are shown graphically in FIGS. 54, 56, 58, and 60, and aretabulated in Table 22, below.

In the animals treated between weeks 9-12, HE stained liver sectionsfrom the Vehicle group exhibited severe micro- and macrovesicular fatdeposition, hepatocellular ballooning and inflammatory cell infiltration(FIG. 53A-B). The PEG-Compound 1 group showed marked improvements in fatdeposition, hepatocellular ballooning and inflammatory cellinfiltration, with significant reduction in NAS compared with theVehicle group (FIG. 53C-D).

In the animals treated between weeks 9-15, the PEG-Compound 1 groupshowed marked improvements in fat deposition, hepatocellular ballooningand inflammatory cell infiltration (FIG. 53E-F), with significantreduction in NAS compared with the Vehicle group (FIG. 53G-H).

NALFD activity score results are shown graphically in FIG. 54A-B foranimals treated between weeks 9-12 and 9-13, respectively, and aresummarized in Table 20. NALFD activity score was significantly decreasedin both treatment groups (p<0.01 and p<0.001 for animals treated between9-12 and 9-15 weeks, respectively).

TABLE 20 Summary of NALFD activity score results. Score components areas shown in Table 21, below. Lobular Hepatocyte NAS SteatosisInflammation ballooning (mean ± Group N 0 1 2 3 0 1 2 3 0 1 2 SD) 12 wks6 1 4 — 1 — 2 4 — — — 6 4.8 ± 1.2 Vehicle 12 wks PEG- 6 6 — — — 1 2 2 12 4 — 2.2 ± 0.8 Compound 1 15 wks 6 1 3 1 1 — 1 5 — — — 6 5.2 ± 1.2Vehicle 15 wks PEG- 14 11 3 — — 6 6 2 — 7 5 2 1.6 ± 1.2 Compound 1

TABLE 21 NALFD activity score components. Item Score Extent Steatosis 0    <5% 1  5-33% 2 >33-66% 3    >66% Hepatocyte Ballooning 0 None 1 Fewballoon cells 2 Many cells/prominent ballooning Lobular Inflammation 0No foci 1 <2 foci/200x 2 2-4 foci/200x  3 >4 foci/200x

In the animals treated between weeks 9-12, sirius red stained liversections from the Vehicle group exhibited collagen deposition in thepericentral region of liver lobule (FIG. 55A-B). There were nosignificant differences in the fibrosis area between the Vehicle groupand the PEG-Compound 1 group (FIG. 55C-D), as summarized in FIG. 56A.

In the animals treated between weeks 9-15, the fibrosis areasignificantly decreased in the PEG-Compound 1 group (FIG. 55E-F)compared with Vehicle group (FIG. 55G-H), as summarized in FIG. 56B(p<0.05).

Representative photomicrographs of the F4/80-immunostained sections areshown in FIG. 57.

In the animals treated between weeks 9-12, F4/80 immunostaining of liversections form the Vehicle group demonstrated accumulation of F4/80+cells in the liver lobule (FIG. 57A-B). There were no significantdifferences in the number and size of F4/80+ cells between the Vehiclegroup and the PEG-Compound 1 group (FIG. 57C-D), as summarized in FIG.58A.

In the animals treated between weeks 9-15, there were no significantdifferences in the number and size of F4/80+ cells between the Vehiclegroup (FIG. 57E-F) and the PEG-Compound 1 group (FIG. 57G-H), assummarized in FIG. 58B.

Representative photomicrographs of the oil red-stained sections areshown in FIG. 59A-H

In the animals treated between weeks 9-12, oil red-stained liversections from the Vehicle group exhibited micro- and macrovesicular fatdeposition in the hepatocytes (FIG. 59A-B). The percentage of fatdeposition area (oil red-positive area) significantly decreased in thePEG-Compound 1 group (FIG. 59C-D) compared with the Vehicle group, assummarized in FIG. 60A (p<0.001).

In the animals treated between weeks 9-15, the percentage of fatdeposition area significantly decreased in the PEG-Compound 1 group(FIG. 59E-F) compared with the Vehicle group (FIG. 59G-H), as summarizedin FIG. 60B (p<0.001).

TABLE 22 Summary of Histological Findings Parameter 12 wks Vehicle 12wks PEG-Compound 1 (mean ± SD) (n = 6) (n = 6) Liver fibrosis area (%)1.17 ± 0.33 0.97 ± 0.51 Inflammation area (%) 3.38 ± 0.70 3.47 ± 1.40Fat deposition area (%) 40.1 ± 4.8  16.2 ± 7.9  Kidney fibrosis area (%)1.70 ± 0.49 1.67 ± 0.92 Parameter 15 wks Vehicle 15 wks PEG-Compound 1(mean ± SD) (n = 6) (n = 14) Liver fibrosis area (%) 2.18 ± 0.14 1.44 ±0.53 Inflammation area (%) 3.983 ± 1.966 4.511 ± 1.597 Fat depositionarea (%) 32.0 ± 9.6  12.5 ± 10.1 Kidney fibrosis area (%) 1.58 ± 0.411.77 ± 0.69

As noted above, PEG-Compound 1 reduced hepatic fat accumulation asassessed by a biochemical assay measuring hepatic triglyceride contentand histology following staining of liver sections with hematoxylin andeosin or oil red O. This anti-steatotic activity of native FGF21 hasbeen reported in the literature to depend on adiponectin in the mouse(see Lin et al., Cell Metab. 17: 779-789 (2013); Holland et al., CellMetab 17: 790-797 (2013), each of which is hereby incorporated byreference in its entirety). Therefore, concentrations of totaladiponectin were measured in terminal serum samples prepared from thetreated mice. Serum adiponectin was measured following themanufacturer's protocol using a commercially available ELISA kit (Alpcocatalog number 47-ADPMS-E01).

Twice weekly administration of 3 mg/kg PEG-Compound 1 statisticallysignificantly increased serum total adiponectin, as compared to thecorresponding vehicle group, at all terminal time-points tested (FIG.62). This result is consistent with the hypothesis that adiponectincontributes to the efficacy of PEG-Compound 1 in the Stelic NASH model.

In summary, treatment for three weeks with PEG-Compound 1 significantlyreduced liver-to-body weight ratio, liver lipid content (triglycerideand cholesterol), fat deposition area and NAS. In addition to theseeffects, treatment for 6 week with PEG-Compound 1 significantly reducedthe whole blood glucose, plasma ALT, and fibrosis area. Moreover,PEG-Compound 1 treatment improved survival rate compared with theVehicle group.

In conclusion, PEG-Compound 1 showed anti-NASH, and anti-fibroticeffects including improved lipid and glucose metabolism.

Example 17 In Vitro Characterization of PEGylated Compound 2 in HumanEmbryonic Kidney Cells Stably Expressing Beta-Klotho

FGF21 utilizes β-klotho as a co-receptor along with FGF receptors forits tissue-specific signaling activity (see, e.g., Kurosu et al., 2007,J. Biol. Chem. 282:26687-26695; Ogawa et al., 2007, Proc. Natl. Acad. ofSci., USA 104:7432-7437, each of which is incorporated by reference inits entirety). FGF21 first binds to β-klotho and the complex can thenactivate FGF receptors to initiate an intracellular signaling cascadethat rapidly activates extracellular signal-regulated kinases 1/2(ERK1/2) over the course of minutes. Over longer times, activated,phosphorylated ERK (pERK) can translocate to the cell nucleus andphosphorylate and activate a number of transcriptional regulatorsincluding a ternary complex factor referred to as E Twenty-Six (ETS)like protein 1 (Elk1). Activated Elk1 forms a complex with serumresponse factor that binds to certain DNA sequences to regulateexpression of certain genes.

Generation of a Cell Line Stably Co-Expressing Human β-Klotho and anElk1-Luciferase Trans-Reporter Construct

The Elk1-luciferase trans-reporting cell line was generated with HEK293cells utilizing Agilent Technologies' PathDetect Elk1 trans-ReportingSystem. This system has a transactivator fusion plasmid that expresses afusion protein of the DNA binding domain from the yeast transcriptionalactivator protein, Gal4, followed by the activation domain of Elk1. Whenthe transcriptional activator, Elk1, is phosphorylated and activated byextracellular signal-regulated kinase (ERK) upon stimulation by FGF21,the fusion-activator protein binds as a dimer to the GAL4 upstreamactivation sequence and activates transcription of the luciferasereporter enzyme. Luciferase expression from the reporter plasmidindicates the activation of the fusion trans-activator protein and,therefore, the presence of the endogenous protein kinase. Accordingly,luciferase activity reflects the activation of ERK in the cell and thecorresponding signal transduction pathways, in this case, activation ofElk1.

HEK293 cells were co-transfected with the fusion trans-activatorplasmid, the reporter plasmid, and a plasmid encoding human β-klotho.The transfected cells were under the selection of G-418 (500 μg/ml) andblasticidin (10 μg/ml). The clones that survived dual antibioticselection were analyzed by stimulating with an FGF21 construct andfollowing Elk1-luciferase activity. HEK Luc Clone2 was selected as thecell line for all future assays.

Cell-Based Elk1-Luciferase Assay

The HEK293 Luc Clone2 cells were seeded in 96-well plates at a densityof 25,000 cells per well. The plates were incubated in a tissue cultureincubator at 37° C. with 5% CO₂ for two days. On the day of the assay, a3-fold serial dilution series of the FGF21 proteins was prepared inphenol red-free DMEM media supplemented with 1× L-glutamine, 10 mMHepes, pH 7.2-7.5, and 10% FBS. The old cell culture growth medium wasremoved. To start the treatment, 50 μl of phenol red-free DMEM mediumwith the testing compounds was added into each well and the plates wereplaced back into the cell culture incubator. At the end of 5 hours ofincubation, an equal volume of luciferase substrate reagent mixture wasadded in each well. The plates were covered and placed on an orbitalshaker for 3 min at 600 rpm. The luminescence was measured on PerkinElmer's Envision 2103 Multiplate reader.

Data Analysis

The raw data from all of the individual replicate data points wereanalyzed with GraphPad Prism 5 software (GraphPad Software Inc., CA)using a non-linear regression analysis. The data were fit using thelog(agonist) against response equation with 4 parameters: Bottom(minimum), Top (maximum), log EC50 (measure of potency), and a variableHill slope. The potency (EC50) was defined as the concentration thatproduced a half-maximal increase in the response above the baseline andwas calculated by the curve-fitting program

Results

Representative concentration-response curves for three FGF21 variants(N-terminally His-tagged Compound 1 (His-Compound 1), PEG-Compound 1 andPEG-Compound 2) and the calculated potency and efficacy values in the 5hours Elk1-luciferase assay are shown in FIG. 63 and Table 23. Thepotencies of the three variants were similar in the 5 hourElk1-luciferase assay (Table 23).

TABLE 23 Potency And Efficacy of FGF21 Variants in the 5 hourElk1-luciferase Assay Potency Efficacy Compound pEC50^(a) (log M)EC50^(b) (nM) Emax^(a) (RLU) % Control^(c) His-Compound 1 8.99 ± 0.10 1.0 (0.8-1.3)  1.90E06 ± 3.9E05 100 PEG-Compound 1 8.44 ± 0.05^(d) 3.6(3.2-4.1)^(d) 1.60E06 ± 3.7E05 84.5 ± 19.8 PEG- Compound 2 8.27 ±0.16^(d) 5.4 (3.7-8.0)^(d) 1.66E06 ± 4.5E05 87.8 ± 23.9 ^(a)Mean ± S.D.^(b)Geometric Mean (95% confidence interval) ^(c)Control was defined asHis-Compound 1 ^(d)P < 0.05 compared to His-Compound 1, ANOVA withTukey-Kramer post-hoc test.

Various Fc-FGF-21 fusion molecules were compared in the cell-basedElk1-luciferase assay. All of the molecules except Compound 180 andCompound 175 exhibited single digit nM potencies or lower and werecomparable to or lower than the measured potency of PEG-Compound 2 (FIG.64 and Table 24 below).

TABLE 24 Geometric Mean EC50 values with 95% confidence intervals. CmpdCmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd Cmpd PEG- 170 171 172173 174 175 176 177 178 179 180 181 Cmpd 2 Geometric Mean (nM) 3.8 1.71.5 1.5 3.3 14.6 1.2 1.8 1.3 2.5 164.8 0.8 5.9 Lower 95% CI of mean 2.81.0 0.6 1.2 1.3 6.1 1.0 1.4 1.0 1.2 92.9 0.6 4.9 Upper 95% CI of mean5.2 2.9 3.6 1.7 8.4 34.8 1.5 2.4 1.6 5.0 291.7 1.1 7.1

Example 18 Effects of FGF-21 Variant Compounds on Adiponectin Levels inC57BL/6J Mice

FGF21 stimulates secretion of adiponectin from adipocytes, and the broaddistribution of adiponectin receptors provides a potential avenue bywhich FGF21 action can be extended to other tissues. Many of themetabolic effects of native FGF21, including its anti-steatoticactivity, have been reported to require adiponectin in the mouse (Lin,Z. et al., Cell Metab. 17: 779-789 (2013); Holland et al., Cell Metab17: 790-797 (2013), each of which is hereby incorporated by reference inits entirety). The present example reports the effects of singleascending doses of PEG-Compound 2 on selected pharmacodynamic markers ofFGF21 activities in normal, non-diabetic C57BL/6J mice.

8 week old male C57BL/6J mice were randomized into the followingtreatment groups (sample size n=10/group): 1) Vehicle, 2) PEG-Compound2, 0.03 mg/kg, 3) PEG-Compound 2, 0.1 mg/kg, 4) PEG-Compound 2, 0.3mg/kg, or 5) PEG-Compound 2, 1.0 mg/kg, and dosed subcutaneously withtheir respective treatment at 5 ml/kg. Following dosing in thenon-fasted state, blood samples were centrifuged and 20 μl plasma wasaliquoted for the quantitative determination of total and high molecularweight (HMW) Adiponectin (Alpco Diagnostics, Salem, N.H., cat#47-ADPMS-E01). Mice were again bled in the non-fasted state 24, 48, 72,and 144 hours following the single acute dose for plasma glucose andadiponectin analyses, and body weight was measured prior to each bleed.

The plasma concentrations of total and high molecular weight (HMW)adiponectin were measured by following the manufacturer's protocol forthe Mouse Total and HMW ELISA kit (Alpco Diagnostics, Salem, N.H.).Plasma exposures of PEG-Compound 2 were determined by ELISA. Statisticalanalysis of all the data was performed using one-way ANOVA followed byDunnett's t-test using the Vehicle group as the control (JMP from SAS,Cary, N.C.). Statistical significance was determined at probability(P)<0.05.

Body weight was determined at baseline (0), 24, 48, 72 and 144 hoursafter the treatment. When converted to percent change from baseline (day0) body weight, a significant dose-dependent decrease was observed inresponse to single SC doses of PEG-Compound 2 (FIG. 65). The percentweight loss effect was maximal at 72 hours post-dose and began to returnto baseline values by day 6, with the response to 1.0 mg/kg dose ofPEG-Compound 2 remaining significantly decreased as compared to that inthe vehicle group. Body weight was significantly decreased compared tothe vehicle treatment group for the 0.3 mg/kg treatment group at 72hours (P<0.001) and for the 1.0 mg/kg treatment group at 48 hours(P<0.05), 72 hours (P<0.0001) and 144 hours (P<0.001).

Plasma total adiponectin concentrations were significantly increasedrelative to vehicle by treatment with 0.1, 0.3 and 1.0 mg/kg doses ofPEG-Compound 2 at 72 and 144 hours post-dose (FIG. 66A). The 0.03 mg/kgdose of PEG-Compound 2 tested did not significantly increase totaladiponectin effects compared to vehicle at any point measured. Plasmatotal adiponectin was significantly increased in the 0.1 mg/kg treatmentgroup at 72 hours (P<0.001) and 144 hours (P<0.01), in the 0.3 mg/kgtreatment group at 72 hours (P<0.01) and 144 hours (P<0.01), and in the1 mg/kg treatment group at 72 hours (P<0.0001) and 144 hours (P<0.0001).

When expressed as percent change from baseline, plasma total adiponectinconcentrations were significantly increased compared to vehicle bytreatment with all doses of PEG-Compound 2 at some of the time-points(FIG. 66B). There was no significant difference in percent change frombaseline in total adiponectin compared to vehicle 24 hours aftertreatment with any dose of PEG-Compound 2, and at 72 hours post-dose,all doses of PEG-Compound 2 produced statistically significant increasescompared to vehicle. The maximal change observed was a 75% increase inchange from baseline adiponectin 144 hours post-dose with 1 mg/kgPEG-Compound 2. Percentage change in plasma total adiponectin wassignificantly increased in the 0.03 mg/kg treatment group at 72 hours(P<0.01), in the 0.1 mg/kg treatment group at 48 hours (P<0.01), 72hours (P<0.0001) and 144 hours (P<0.001), in the 0.3 mg/kg treatmentgroup at 72 hours (P<0.0001) and 144 hours (P<0.0001), and in the 1.0mg/kg treatment group at 48 hours (P<0.01), 72 hours (P<0.0001) and 144hours (P<0.0001).

Plasma HMW adiponectin concentrations were significantly increasedcompared to vehicle by treatment with the 0.1 and 1.0 mg/kg doses ofPEG-Compound 2, while the 0.03 and 0.3 mg/kg dose of PEG-Compound 2 didnot significantly increase HMW adiponectin compared to vehicle at anypoint measured (data not shown).

When measured at the end of the study 144 hours after treatment, plasmaconcentrations of PEG-Compound 2 increased dose-dependently for both theactive, carboxy (C)-terminal intact and the total (intact andproteolyzed) forms of the molecule Table 25.

TABLE 25 Plasma concentrations of total and C-terminal intactPEG-Compound 2 after single SC doses Form of PEG- Dose (mg/kg) Compound2 0.03 0.1 0.3 1.0 Total (ng/ml)  1.5 ± 0.21  12 ± 0.89 39 ± 3.2 222 ±15  C-terminal Intact 0.40 ± 0.11 5.2 ± 0.38 19 ± 1.4 135 ± 8.6 (ng/ml)Total and C-terminal intact PEG-Compound 2 in C57BL/6J mouse plasma 144hours post SC dose. All values are Mean ± S.E.M.

A similar protocol was followed with molecules PEG-Compound 20 Compound105, Compound 112, Compound 182, Compound 171, Compound 170, Compound181, and PEG-Compound 1, with 9-11 week old C58BL/6J mice, except thattotal adiponectin was only measured at baseline, 3 days (72 hours) and 6days (144 hours) post-dose. The compounds were expressed in E. coliexcept Compound 181, which was expressed in HEK cells. All compoundssignificantly increased plasma total adiponectin compared to vehicle onDay 3 and/or Day 6 at certain doses tested (see FIG. 67A-E).

At dosages of 0.3 mg/kg and 1 mg/kg, PEG-Compound 2 and PEG-Compound 20each resulted in significant increases in plasma total adiponectin 6days post-dose (P<0.001 for 1 mg/kg of either compound, P<0.01 for 0.3mg/kg PEG-Compound 2, and P<0.05 for 0.3 mg/kg PEG-Compound 20) (FIG.67A).

At a dosage of 54.3 nmol/kg, the percentage change from baseline inplasma total adiponectin was significantly increased by PEG-Compound 2,Compound 105, and Compound 112 (as compared to vehicle-treated controls)on Days 3 and 6 (P<0.001 for all data points except PEG-Compound 2 atday 3, P<0.05) (FIG. 67B). Additionally, the 5.43 nmol/kg dose ofCompound 112 significantly increased the percentage change in plasmatotal adiponectin, as compared to vehicle-treated controls, on Day 3only (P<0.05) (FIG. 67B).

The percentage change from baseline in plasma total adiponectin wassignificantly increased by Compound 182 at 18.1 nmol/kg (P<0.01 on bothDays 3 and 6) and 54.3 nmol/kg dose (P<0.001 and P<0.05, respectively,on Days 3 and 6) (FIG. 67C). The increase in the percentage change inplasma total adiponectin for the 5.43 nmol/kg dose of Compound 182 wassignificant on Day 6 only (P<0.05). Additionally, PEG-Compound 2 (54.3nmol/kg dose) significantly increased the percentage change in plasmatotal adiponectin on both Day 3 and Day 6 (P<0.001 on both days) (FIG.67C).

The percentage change from baseline in plasma total adiponectin wassignificantly increased by both Compound 171 and Compound 181 at bothDay 3 and Day 6 in a dose-dependent manner, compared to thevehicle-treated control (P<0.001 for all data points except P<0.01 forthe 5.43 nmol/kg dose of Compound 181 at Day 6) (FIG. 67D).Additionally, PEG-Compound 2 (54.3 nmol/kg dose) significantly increasedthe percentage change in plasma total adiponectin on both Day 3 and Day6 (P<0.001 on both days) (FIG. 67D).

The percentage change from baseline in plasma total adiponectin wassignificantly increased by Compound 170 on Days 3 and 6 at 18.1 nmol/kg(P<0.01, both days) and 54.3 nmol/kg (P<0.05 on Day 3 and P<0.001 on Day6); the increase in the percentage change in plasma total adiponectinfor the 5.43 nmol/kg dose was significant on Day 6 only (P<0.05) (FIG.67E). Additionally, PEG-Compound 2 (54.3 nmol/kg dose) significantlyincreased the percentage change in plasma total adiponectin on both Day3 and Day 6 (P<0.001 on both days) (FIG. 67E).

The percentage change from baseline in plasma total adiponectin wassignificantly increased by Compound 1 on Days 3 and 6 at a dose of 10mg/kg (P<0.001, both days). Additionally, PEG-Compound 2 (1 mg/kg)significantly increased the percentage change in plasma totaladiponectin on Day 6 (P<0.001) (FIG. 67F).

Example 19 Effects on Diabetic Kidney Disease in Db/Db Mice

Uninephrectomized (unix) db/db mice are a rodent model of diabetickidney disease (DKD) in which the surgical removal of one kidneyexacerbates and accelerates the renal injury and dysfunction thatresults from type 2 diabetes (Ninichuk et al., Eur J Med Res 12:351-355(2007), which is hereby incorporated by reference in its entirety).

4-Week Study

In this study, db/db mice at 8 wk of age were subjected to leftnephrectomy under anesthesia with 5% isoflurane to hasten thedevelopment of diabetic nephropathy. Three weeks after uninephrectomy,groups of fourteen uninephrectomized mice were distributed to eitherPEG-Compound 2 or vehicle treatment, based on randomization with urinealbumin to creatinine ratio (uACR), blood glucose, and body weight inthat order. A group of db/m lean mice (n=12) was assigned as thenon-diabetic normal control. Treatment with either PEG-Compound 2 orvehicle in the uninephrectomized db/db mice started at three weeks postsurgery. 0.15 mg/kg PEG-Compound 2 was administered twice weekly (BIW)by subcutaneous (SC) injection in a vehicle containing 250 mM Sucrose,20 mM Tris, pH 8.3. The baseline blood samples were collected viaretro-orbital bleeding under anesthesia with 5% isoflurane. Multiplethree-hour spot urine collections for measurements of urine glucose,albumin and creatinine were obtained from each mouse at baseline, andfollowing 2, 3, and 4 weeks of treatment.

Mice were sacrificed under isoflurane anesthesia following four weeks oftreatment. Statistical analysis of the data was performed using one-wayanalysis of variance (ANOVA) followed by Dunnett's test, unlessotherwise indicated (JMP statistical analysis software, SAS, Cary,N.C.). Statistical significance was determined at probability (P)<0.05.Four mice in the unix db/db Vehicle group were excluded from dataanalyses (except for food and water consumption) because they exhibitedsevere pyelonephritis.

The PEG-Compound 2 and Vehicle groups exhibited similar levels of plasmaglucose (765±42 vs 815±46, P>0.05) at baseline prior to treatment.Following four weeks of treatment at the end of the study, plasmaglucose in the PEG-Compound 2 group was 23% lower than that of theVehicle group (485±39 vs 629±43, P<0.05) (see Table 26).

TABLE 26 PEG-Compound 2 Treatment Lowered Blood Glucose (mg/dL)^(a).Baseline Blood Terminal Blood Group Glucose Glucose Lean 194.9 ± 6.4  181.0 ± 19.5  Unix db/db, Vehicle 814.8 ± 45.6^(b) 628.6 ± 39.0^(b) Unixdb/db, PEG-Compound 2 628.6 ± 39.0^(b) 485.2 ± 42.5^(c) ^(a)Data areexpressed as Mean ± S.E.M. ^(b)P < 0.05, disease (unix db/db, Vehicle, n= 9) versus normal (db/m lean, n = 10). ^(c)P < 0.05, PEG-Compound 2 (n= 14) versus Vehicle (n = 9).

After the initiation of treatment with PEG-Compound 2, urine glucoselevels decreased progressively. Urinary glucose levels of thePEG-Compound 2 group showed significant reductions of 71%, 73%, and 84%with 2, 3 and 4 weeks of treatment, respectively, when compared to theVehicle group (all P<0.05; Table 5).

TABLE 27 PEG-Compound 2 Reduced Urine Glucose (mg/dL)^(a). Group 2 wksdosing 3 wks dosing 4 wks dosing Lean 17 ± 3  12 ± 1  40 ± 5  Unixdb/db, Vehicle 8719 ± 694^(b) 8550 ± 204^(b) 9796 ± 378^(b) Unix db/db,PEG- 2537 ± 818^(c) 2284 ± 715^(c) 1565 ± 410^(c) Compound 2 % reduction71% 73% 84% ^(a)Data expressed as Mean ± S.E.M. ^(b)P < 0.05, disease(unix db/db, Vehicle, n = 9-10) versus normal (db/m lean, n = 10-12).^(c)P < 0.05, PEG-Compound 2 (n = 13-14) versus Vehicle (n = 9-10).

Microalbuminuria (Urine Albumin to Creatinine Ratio (uACR)>30 μg/mg) hasbeen reported in the literature to be one of the most sensitive andspecific indicators of early nephropathy in diabetic patients (see Chaeet al., J Korean Med Sci. 2012; 27: 784-787, which is herebyincorporated by reference in its entirety). Microalbumin was measuredfrom 3 hrs of spot urine collection and the values were normalized tourine creatinine. Nondiabetic db/m lean mice had mean uACR valuesranging from 21 to 37 μg/mg during the study course (FIG. 68 and Table28).

During the study, uACR in unix db/db mice increased significantly from23- to 62-fold (all P<0.05) compared to non-diabetic, lean db/m mice(FIG. 68 and Table 28). The uACR of the Vehicle-treated group progressedfrom 838±159 μg/mg at baseline to 1341±284 μg/mg after 4 weeks (Table28), consistent with progressive renal injury.

Compared to the Vehicle group, PEG-Compound 2-treated unix db/db miceshowed significant decreases in uACR values of 49%, 53% and 66% with 2,3, and 4 weeks of treatment, respectively (all P<0.05; FIG. 68). TheuACR values after PEG-Compound 2 treatment were lower than thosemeasured at the pre-treatment baseline (Table 28).

TABLE 28 PEG-Compound 2 Reduced uACR (μg/mg)^(a). Groups Baseline 2 wksdosing 3 wks dosing 4 wks dosing Lean 36.7 ± 2.6  26.3 ± 4.3  21.4 ±2.7  30.1 ± 2.8  Unix db/db,  838.2 ± 159.4^(b) 1060.2 ± 256.9^(b)1319.9 ± 282.3^(b) 1341.3 ± 283.5^(b) Vehicle Unix db/db, PEG- 865.8 ±170.8  538.5 ± 107.5^(c)  616.1 ± 145.2^(c) 460.6 ± 92.7^(c) Compound 2

Kidney wet weight reflects the status of renal hypertrophy due tohyper-filtration in diabetes mellitus. Mean kidney weight wassignificantly greater in unix db/db mice compared to non-diabetic leanmice, with a 59% increase in the kidney weight of unix db/db micetreated with vehicle. Treatment with PEG-Compound 2 for four weekssignificantly inhibited the kidney weight increase in the diabetic mice(286±6.3 versus 253±7.4, Vehicle versus PEG-Compound 2, P<0.05).

8-Week Study

At 8 wk of age, db/db mice were subjected to left nephrectomy under 3%isoflurane in oxygen to accelerate the development of diabeticnephropathy. Three weeks after uninephrectomy, groups of fourteenuninephrectomized mice were blocked, randomized, and distributed toeither PEG-Compound 2 at 0.15 mg/kg or 0.015 mg/kg or the vehicletreatment, based on urine albumin to creatinine ratio (uACR), bloodglucose, and body weights in that order. A group of db/m lean mice(n=11) was assigned as the non-diabetic normal control. Twice weeklysubcutaneous treatment with PEG-Compound 2 at either 0.15 or 0.015 mg/kgor the vehicle in the unix db/db mice started at three weeks postsurgery. Mice were sacrificed under 3% isoflurane in oxygen followed bycervical dislocation after eight weeks of treatment. Kidneys wereexcised, decapsulated, and weighed.

The 0.015 mg/kg dose group did not show any significant blood glucoselowering or improvement of renal parameters. The 0.15 mg/kg dose ofPEG-Compound 2 significantly lowered blood and urine glucose levels, andreduced albuminuria by ˜50%, compared to that in vehicle-treated mice(all P<0.05). A cluster of inflammatory and fibrotic genes wassignificantly up-regulated in the kidneys of uninephrectomized db/dbmice versus lean mice, and treatment with PEG-Compound 2 at 0.15 mg/kgsignificantly reduced expression of these genes toward levels seen inlean db/m control mice. Mean kidney wet weight was also significantlyhigher in uninephrectomized db/db mice compared to that of lean mice,and treatment with PEG-Compound 2 at 0.15 mg/kg for 57 dayssignificantly reduced mean kidney wet weight as compared to the vehicle.

Sections from the middle ⅓ of the kidney were stained with hematoxylinand eosin for the histological analysis. Kidney lesions were gradedaccording to the criteria defined in the notes to Table 29. All unixdb/db mice exhibited mesangial glomerulopathy, which is the expectedphenotype of this model (Table 29). Treatment with PEG-Compound 2 at0.15 mg/kg, but not at 0.015 mg/kg, appeared to reduce the severity ofthe mesangial glomerulopathy. A few unix db/db mice exhibitedtubular-interstitial lesions secondary to glomerulopathy, which isconsistent with the underlying pathogenesis of the model.

These observations provide evidence that PEG-Compound 2 at 0.15 mg/kg isrenoprotective in the setting of chronic type 2 diabetes.

TABLE 29 PEG-Compound 2: Incidence and Grade of Kidney Lesions.Treatment (mg/kg) 0 (vehicle control) 0.015 0.15 No. of Mice LocaleGrade 9 8 9 Glomeruli^(a) 9 8 9 1 1 1 6 2 5 2 3 3 3 5 — Tubules/ 3 3 4Interstitium^(b) 1 2 2 4 2 1 1 — Pelvis^(c) 2 1 2 1 2 — 1 2 — 1 1^(a)Glomerular mesangial expansion is focal or diffuse, and segmental orglobal. Grade 2 is based on an estimate of >50% glomeruli with mesangialexpansion, and/or an additional mesangial, capillary, epithelial, orcapsular lesion in at least 1 glomerulus; grade 3 is based on more thanone of these additional glomerular lesions. ^(b)Tubular lesions includedilatation, atrophy, and/or hyperplasia; interstitial lesions typicallyare associated with glomerular lesions and characterized by minimalinflammation or localized peri-tubular fibrosis. Grade 1 is focal; grade2 is multifocal with 2 to 4 foci. ^(c)Suppurative inflammation; grade 1is based on inflammation limited to the pelvic epithelium; grade 2 isbased on extension into the medulla or peri-pelvic cortex.

Example 20 Effects on Cardiac Hypertrophy and Cardiac Fibrosis in theIsoproterenol Infused Balb/C Mouse

The isoproterenol (Iso)-infused Balb/c mouse is a rodent model ofcardiac fibrosis and hypertrophy.

Male Balb/c mice at 10 to 12 wks were randomized based on body weightprior to drug administration. Iso or vehicle (0.02% ascorbic acid insaline) was administered subcutaneously via osmotic mini-pump for 3 wks.Mice were concurrently dosed twice weekly (BIW) with 0.5 mg/kgPEG-Compound 2 or Vehicle over the course of 3 wks. The animals werethen euthanized and the hearts were removed at the great vessels andblotted lightly to remove any blood in the ventricles and then weighed.From the remaining 24 mice in each group, the entire heart was frozen ondry ice and stored at ≦−70° C. for hydroxyproline (HP) analysis.

Cardiac HP content (Cardiac fibrosis endpoint) analysis was carried outusing total collagen assay kit according to the manufacturer'sinstructions (Quickzyme Biosciences, Netherlands).

At the end of the 3-wk in-life phase, whole heart HP content wasmeasured. Normalized HP content increased 114% (P<0.05) in theIso+Vehicle group (21.35±0.81 μg/mg) compared to the Sham+Vehicle group(9.96±0.38 μg/mg). This indicated that cardiac fibrosis had beenestablished in response to the Iso infusion in the Balb/c mice. CardiacHP content in mice receiving Iso+PEG-Compound 2 (0.5 mg/kg) (15.91±0.49μg/mg) was significantly less than in the Iso+Vehicle animals (FIG. 69and Table 30). Thus, when administered in a preventative mode,PEG-Compound 2 (0.5 mg/kg) reduced the extent of cardiac hypertrophy andfibrosis.

TABLE 30 Cardiac HP content at week 3 of treatment with isoproterenoland PEG-Compound 2. Data represent whole heart HP content normalized toprotein. All values are Mean ± S.E.M. Sham + Iso + Iso + PEG- Vehicle(n) Vehicle (n) Cmpd 2 (n) Hydroxyproline/ 9.96 ± 0.38 21.35 ± 0.8115.91 ± 0.49 protein (μg/mg) (20) (19)* (20)** *P < 0.05, vs Sham +Vehicle, **P < 0.05, vs Iso + Vehicle (One-way ANOVA followed byBonferroni's test).

An intervention study was performed to determine if 0.5 mg/kgPEG-Compound 2 could delay progression or reverse pre-establishedcardiac hypertrophy and fibrosis in Balb/c mice that had already beeninfused with Iso for 1 wk prior to initiating 3 wk of treatment with thePEG-FGF21 variant. PEG-Compound 2 (0.5 mg/kg) intervention resulted in a24% reduction in left ventricular (LV) mass (a cardiac hypertrophyendpoint assessed by echocardiography). This observation was associatedwith a significant, 14% loss of body weight and 23% elevation ofcirculating levels of adiponectin. No significant difference wasobserved between the Iso+Vehicle and Iso+PEG-Compound 2 groups incardiac fibrosis (HP content) or systolic cardiac function (measured byechocardiograph).

Example 21 A Randomized, Placebo-Controlled, Single and MultipleAscending Dose Study to Evaluate the Safety, Tolerability,Pharmacokinetics and Pharmacodynamics of PEG-Compound 2 in HealthySubjects

The study consists of three parts: Part A: Single Ascending Dose (SAD;Part B: Multiple Ascending Dose (MAD); and Part C: Multiple AscendingDose in Japanese subjects (J-MAD). PEG-Compound 2 or placebo isadministered by subcutaneous injection as a blinded treatment. Thesubjects in Part A receive a single dose of blinded treatment and thesubjects in Parts B and C receive multiple doses of blinded treatment.

The starting dose of 0.6 mg PEG-Compound 2 in SAD is selected. A topdose of 60 mg in SAD and a 60 mg weekly dosing for MAD are also selectedto explore the higher end of the projected efficacious dose range. Asubject may receive a dose of 0.6 mg, 2 mg, 6 mg, 20 mg, 40 mg, or up to60 mg PEG-Compound 2 in the SAD arm. In MAD arms, a subject may receivea weekly dose of 2 mg, 6 mg, 20 mg, 40 mg, or 60 mg (QW or BIW).

Healthy male and female subjects aged 21-55 years with BMI between 30and 40 (Parts A and B) or between 20 and 35 (Part C) may be included.

Primary Endpoints:

The objective (to assess the safety and tolerability of single andmultiple subcutaneous doses of PEG-Compound 2 in healthy subjects) willbe measured by the following safety endpoints: incidence of AEs, seriousAEs, and events of special interest including injection site assessment,AEs leading to discontinuation, and death as well as markedabnormalities in clinical laboratory tests, vital sign measurements,ECGs, physical examinations occurring up to 30 days after the last doseof study medication.

Secondary Endpoints:

Part A: Single-dose pharmacokinetic parameters (Cmax, Tmax, AUC(0-T),AUC(INF), T-HALF, and CLT/F) derived from the C-Terminal intactPEG-Compound 2 serum concentration versus time data, on the day ofsingle dose treatment and for up to 4 weeks washout after the singledose.

Part B and C: Multiple-dose pharmacokinetic parameters (Cmax, Tmax,AUC(TAU) and the accumulation index (AI)) derived from PEG-Compound 2serum concentration versus time data following first dose and last dose.T-HALF will be calculated only for the last dose. PEG-Compound 2 troughserum concentration (CTrough) will be obtained on selected days duringthe course of 22 days of treatment and for up to 4 weeks after the lastdose.

Example 22 Assessment of PEG-Compound 1, PEG-Compound 2, and Compound170 in a Mouse Model of NASH

Three FGF-21 variants, PEG-Compound 1, PEG-Compound 2, and Compound 170,were tested in the Stelic Institute's 2-hit model of NASH, describedabove in Example 13. NASH was induced in 40 male mice by a singlesubcutaneous injection of 200 μg streptozotocin solution 2 days afterbirth and feeding with high fat diet (HFD; 57% fat by kcal) after 4weeks of age (“STAM mice”). The mice were administered 3 mg/kgPEG-Compound 1 (n=8), 0.5 mg/kg PEG-Compound 2 (n=8), 2.5 mg/kg Compound170 (n=8), or vehicle (20 mM Tris/250 mM sucrose, pH 8.3) (n=8), bysubcutaneous route twice per week from 7- to 9-weeks of age. These micewere sacrificed at 9-weeks of age for various analyses. Another group ofeight mice served as the baseline control and were sacrificed at 7-weeksof age.

Concentrations of total and high molecular weight (HMW) adiponectin weremeasured in terminal serum samples prepared from the mice using acommercially available ELISA kit (Alpco catalog number 47-ADPMS-E01)following the manufacturer's protocol. Compared to the Vehicle group,All 3 FGF21 variants significantly increased serum total adiponectin(FIG. 70A) (p<0.0001 for PEG-Compound 1, PEG-Compound 2, and Compound170), serum HMW adiponectin (FIG. 70B) (p<0.005, PEG-Compound 1; andp<0.0001, PEG-Compound 2, and Compound 170), and the ratio of HMW/totaladiponectin (FIG. 70C) (p<0.05, PEG-Compound 1; and p<0.01, PEG-Compound2, and Compound 170).

Body weight, liver weight, liver triglyceride, liver cholesterol, andplasma glucose, triglycerides, and ALT levels were determined. All threeFGF21 variants significantly reduced body weight (FIG. 72A), liverweight (FIG. 72B), liver-to-body weight ratio (FIG. 72C), livertriglyceride (FIG. 72D), and plasma ALT (FIG. 72E). PEG-Compound 2 andCompound 170 significantly reduced liver cholesterol (FIG. 72F), andPEG-Compound 2 significantly reduced plasma triglycerides (FIG. 72G). Nocompound significantly decreased plasma cholesterol or glucose (data notshown) in this study.

Example 23 Assessment of PEG-Compound 2 and Compound 170 During 3 Weeksof BIW Dosing in Ob/Ob Mice

Pegylated Compound 2 and Compound 170 were evaluated in a 21-dayrepeated dosing study. The results demonstrate that both compoundscaused reductions in liver weight and dose-dependent reductions inhepatocyte steatosis in the left lateral lobe of the liver, as comparedto vehicle.

Male ob/ob mice (Jackson Laboratories, Bar Harbor, Me.) at 8 weeks ofage were randomized into 5 groups as described in Example 12. The micewere administered vehicle, Pegylated Compound 2 and Compound 170subcutaneously twice weekly (BIW) as follows (n=12 for each group): 1)Vehicle (250 mM sucrose/20 mM Tris, pH 8.3); 2) Pegylated Compound 2,8.14 nmol/kg (0.15 mg/kg); 3) Compound 170, 8.14 nmol/kg; 4) PegylatedCompound 2, 81.45 nmol/kg (1.5 mg/kg); and 5) Compound 170, 81.45nmol/kg. Body weight, plasma glucose, triglycerides, insulin,nonesterified fatty acids (NEFA), β-OH-butyrate, total adiponectin, andhigh molecular weight (HMW) adiponectin were determined throughout the21 day dosing period in the fed state. Glycated hemoglobin (HbA1c) inwhole blood was determined at study start and termination. Liver weightand liver triglycerides were determined at study termination. Liverhistology was performed with the left lateral lobe of the liver toevaluate hepatocyte steatosis. The tissue was fixed in 10% bufferedformalin and processed for histology. Sections of paraffin-embeddedtissues were stained with hematoxylin and eosin using standard methods.The first 6 mice in each treatment group were evaluated (total=30 mice).Hepatocyte steatosis (micro- and macro-vesicular fatty change) wasmostly limited to zones II/III in control mice; this degree of steatosiswas arbitrarily assigned a grade (score) 2. Other samples were graded ina blinded manner (a grade 0 assigned to the lowest degree of steatosisobserved). In addition, lipid-laden perisinusoidal cells, presumablystellate (Ito) cells, were observed in mice treated with eitherPEG-Compound 2 or Compound 170, but not Vehicle in which the Ito cellswere obscured by hepatocyte steatosis. The abundance of the Ito cellswas evaluated in a blinded manner (without counting) and each sample wasarbitrarily graded to provide a semi-quantitative assessment (with ascore of 3 indicating the highest abandunce).

Both doses of both compounds significantly reduced fed glucose tonormoglycemic levels (data not shown), and significantly reduced plasmainsulin and HbA1c (data not shown). Significant increases in highmolecular weight (HMW) adiponectin vs. vehicle were observed from Day 8through Day 21, with a trend for dose-dependence, while plasma totaladiponectin demonstrated some small, but inconsistent increases vs.vehicle (data not shown). Plasma NEFA and β-OH-butyrate both increasedwith both compounds (especially at the high dose) vs. vehicle (data notshown), suggesting increased fatty acid oxidation. Significant decreasesin both absolute liver weight and liver weight corrected for body weightwere observed on Day 21 in all treatment groups (FIGS. 71A and 71B).Specifically, in all four groups treated with FGF-21 variants, absoluteliver weight was significantly decreased (p<0.0001) relative to vehiclecontrol, and the higher dose of PEG-Compound 2 significantly decreasedabsolute liver weight relative to the lower dose of that compound(p<0.05) (FIG. 71A). Additionally, in all four groups treated withFGF-21 variants, the ratio of liver weight to body weight wassignificantly decreased (p<0.0001) relative to vehicle control (FIG.71B). A dose-dependent reduction in the severity of hepatocyte steatosiswas observed in mice treated with either PEG-Compound 2 or Compound 170compared to vehicle (Table 31). Lipid-laden perisinusoidal cells(presumably Ito or hepatic stellate cells) were observed in sectionsfrom PEG-Compound 2 and Compound 170 treated mice (Table 32), but theycould not be visualized in sections from Vehicle-treated mice due tointerference from hepatocyte steatosis. A dose-dependent reduction inthe severity of Ito cell prominence was observed in sections from micetreated with either PEG-Cmpd 2 or Compound 170.

TABLE 31 Hepatocyte steatosis (Fatty change) Incidence and HistologyScores in ob/ob mice PEG-Cmpd 2 Cmpd 170 PEG-Cmpd 2 Cmpd 170 Vehicle(8.14 nmol/kg) (8.14 nmol/kg) (81.45 nmol/kg) (81.45 nmol/kg Score No. 66 6 6 6 0 — — 1 6 6 1 — 6 5 — — 2 6 — — — —

TABLE 32 Perisinusoidal Lipid-laden Cell Incidence and Histology Scoresin ob/ob mice. PEG-Cmpd 2 Cmpd 170 PEG-Cmpd 2 Cmpd 170 Vehicle (8.14nmol/kg) (8.14 nmol/kg) (81.45 nmol/kg) (81.45 nmol/kg Score No. 6 6 6 66 0 ND — — 1 — 1 ND — 1 4 1 2 ND — — 1 5 3 ND 6 5 — —

1-89. (canceled)
 90. A modified FGF-21 polypeptide comprising apolypeptide having at least 96% amino acid sequence identity to theamino acid sequence of SEQ ID NO:
 202. 91. The modified FGF-21polypeptide of claim 90, wherein said modified FGF-21 polypeptidecomprises a polypeptide having at least 97% amino acid sequence identityto the amino acid sequence of SEQ ID NO:
 202. 92. The modified FGF-21polypeptide of claim 91, wherein said modified FGF-21 polypeptidecomprises a polypeptide having at least 98% amino acid sequence identityto the amino acid sequence of SEQ ID NO:
 202. 93. The modified FGF-21polypeptide of claim 92, wherein said modified FGF-21 polypeptidecomprises a polypeptide having at least 99% amino acid sequence identityto the amino acid sequence of SEQ ID NO:
 202. 94. The modified FGF-21polypeptide of claim 90, wherein said modified FGF-21 polypeptide islinked to a half-life extending moiety.
 95. The modified FGF-21polypeptide of claim 94, wherein said half-life extending moietycomprises a poly(ethylene glycol).
 96. The modified FGF-21 polypeptideof claim 95, wherein said poly(ethylene glycol) has an average molecularweight between 10 kDa and 40 kDa.
 97. The modified FGF-21 polypeptide ofclaim 95, wherein said poly(ethylene glycol) has an average molecularweight of about 30 kDa.
 98. The modified FGF-21 polypeptide of claim 94,wherein said modified FGF-21 polypeptide is linked to a half-lifeextending moiety via a para-acetyl-L-phenylalanine in the modifiedFGF-21 polypeptide.
 99. A composition comprising the modified FGF-21polypeptide of claim 90 and a pharmaceutically acceptable carrier orexcipient.
 100. A modified FGF-21 polypeptide comprising a polypeptidehaving the amino acid sequence of SEQ ID NO: 202, wherein said modifiedFGF-21 polypeptide is linked to a half-life extending moiety via thepara-acetyl-L-phenylalanine in the modified FGF-21 polypeptide.
 101. Themodified FGF-21 polypeptide of claim 100, wherein the half-lifeextending moiety comprises a water soluble polymer.
 102. The modifiedFGF-21 polypeptide of claim 100, wherein the half-life extending moietycomprises a poly(ethylene glycol).
 103. The modified FGF-21 polypeptideof claim 102, wherein said poly(ethylene glycol) has an averagemolecular weight between 10 kDa and 40 kDa.
 104. The modified FGF-21polypeptide of claim 102, wherein the poly(ethylene glycol) has anaverage molecular weight between 20 kDa and 40 kDa.
 105. A compositioncomprising the modified FGF-21 polypeptide of claim 100 and apharmaceutically acceptable carrier or excipient.
 106. A modified FGF-21polypeptide comprising a polypeptide having the amino acid sequence ofSEQ ID NO: 202, wherein said modified FGF-21 polypeptide is linked to ahalf-life extending moiety comprising a poly(ethylene glycol) via thepara-acetyl-L-phenylalanine in the modified FGF-21 polypeptide.
 107. Themodified FGF-21 polypeptide of claim 106, wherein said poly(ethyleneglycol) has an average molecular weight between 10 kDa and 40 kDa. 108.The modified FGF-21 polypeptide of claim 106, wherein thepara-acetyl-L-phenylalanine in the modified FGF-21 polypeptide is linkedto the half-life extending moiety through an oxime linkage.
 109. Acomposition comprising the modified FGF-21 polypeptide of claim 106 anda pharmaceutically acceptable carrier or excipient.